Immunogenic compositions for streptococcus pyogenes

ABSTRACT

The invention includes a GAS antigen, GAS 40, which is particularly suitable for use either alone or in combinations with additional GAS antigens, such as GAS 117, GAS 130, GAS 277, GAS 236, GAS 40, GAS 389, GAS 504, GAS 509, GAS 366, GAS 159, GAS 217, GAS 309, GAS 372, GAS 039, GAS 042, GAS 058, GAS 290, GAS 511, GAS 533, GAS 527, GAS 294, GAS 253, GAS 529, GAS 045, GAS 095, GAS 193, GAS 137, GAS 084, GAS 384, GAS 202, and GAS 057.

All documents cited herein are incorporated by reference in theirentirety.

This application is a division of Ser. No. 12/715,912 filed on Mar. 2,2010, which is a continuation of Ser. No. 10/565,126, now U.S. Pat. No.7,709,009, which is a national stage entry of PCT/US04/24868 filed onJul. 30, 2004, which claims the benefit and incorporates by reference intheir entireties Ser. No. 60/491,822 filed on Jul. 31, 2003 and Ser. No.60/541,565 filed on Feb. 3, 2004.

This application incorporates by reference the contents of a 436 kb textfile created on Mar. 2, 2012 and named “PAT051962sequencelisting.txt,”which is the sequence listing for this application.

FIELD OF THE INVENTION

This invention is in the fields of immunology and vaccinology. Inparticular, it relates to antigens derived from Streptococcus pyogenesand their use in immunisation. All documents cited herein areincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Group A streptococcus (“GAS”, S. pyogenes) is a frequent human pathogen,estimated to be present in between 5-15% of normal individuals withoutsigns of disease. When host defences are compromised, or when theorganism is able to exert its virulence, or when it is introduced tovulnerable tissues or hosts, however, an acute infection occurs. Relateddiseases include puerperal fever, scarlet fever, erysipelas,pharyngitis, impetigo, necrotising fasciitis, myositis and streptococcaltoxic shock syndrome.

GAS is a gram positive, non-sporeforming coccus shaped bacteria thattypically occurs in chains or in pairs of cells. Although S. pyogenesmay be treated using antibiotics, a prophylactic vaccine to prevent theonset of disease is desired. Efforts to develop such a vaccine have beenongoing for many decades. While various GAS vaccine approaches have beensuggested and some approaches are currently in clinical trials, to date,there are no GAS vaccines available to the public.

It is an object of the invention to provide further and improvedcompositions for providing immunity against GAS disease and/orinfection. The compositions preferably include GAS 40, a GAS virulencefactor identified by Applicants, which is particularly suitable for usein vaccines. In addition, the compositions are based on a combination oftwo or more (e.g. three or more) GAS antigens.

SUMMARY OF THE INVENTION

Applicants have discovered a group of thirty GAS antigens that areparticularly suitable for immunisation purposes, particularly when usedin combinations. In addition, Applicants have identified a GAS antigen(GAS 40) which is particularly immunogenic used either alone or incombinations with additional GAS antigens.

The invention therefore provides an immunogenic composition comprisingGAS 40 (including fragments thereof or a polypeptide having sequenceidentity thereto). A preferred fragment of GAS 40 comprises one or morecoiled-coil regions. The invention further includes an immunogeniccomposition comprising a combination of GAS antigens, said combinationconsisting of two to ten GAS antigens, wherein said combination includesGAS 40 or a fragment thereof or a polypeptide having sequence identitythereto. Preferably, the combination consists of three, four, five, six,or seven GAS antigens. Still more preferably, the combination consistsof three, four, or five GAS antigens.

The invention also provides an immunogenic composition comprising acombination of GAS antigens, said combination consisting of two tothirty-one GAS antigens of a first antigen group, said first antigengroup consisting of: GAS 117, GAS 130, GAS 277, GAS 236, GAS 40, GAS389, GAS 504, GAS 509, GAS 366, GAS 159, GAS 217, GAS 309, GAS 372, GAS039, GAS 042, GAS 058, GAS 290, GAS 511, GAS 533, GAS 527, GAS 294, GAS253, GAS 529, GAS 045, GAS 095, GAS 193, GAS 137, GAS 084, GAS 384, GAS202, and GAS 057. These antigens are referred to herein as the ‘firstantigen group’. Preferably, the combination of GAS antigens consists ofthree, four, five, six, seven, eight, nine, or ten GAS antigens selectedfrom the first antigen group. Preferably, the combination of GASantigens consists of three, four, or five GAS antigens selected from thefirst antigen group.

GAS 39, GAS 40, GAS 57, GAS 117, GAS 202, GAS 294, GAS 527, GAS 533, andGAS 511 are particularly preferred GAS antigens. Preferably, thecombination of GAS antigens includes either or both of GAS 40 and GAS117. Preferably, the combination includes GAS 40.

Representative examples of some of these antigen combinations arediscussed below.

The combination of GAS antigens may consist of three GAS antigensselected from the first antigen group. Accordingly, in one embodiment,the combination of GAS antigens consists of GAS 40, GAS 117 and a thirdGAS antigen selected from the first antigen group. Preferredcombinations include GAS 40, GAS 117 and a third GAS antigen selectedfrom the group consisting of GAS 39, GAS 57, GAS 202, GAS 294, GAS 527,GAS 533, and GAS 511.

In another embodiment, the combination of GAS antigens consists of GAS40 and two additional GAS antigens selected from the first antigengroup. Preferred combinations include GAS 40 and two GAS antigensselected from the group consisting of GAS 39, GAS 57, GAS 117, GAS 202,GAS 294, GAS 527, GAS 533, and GAS 511. In another embodiment, thecombination of GAS antigens consists of GAS 117 and two additional GASantigens selected from the first antigen group.

The combination of GAS antigens may consist of four GAS antigensselected from the first antigen group. In one embodiment, thecombination of GAS antigens consists of GAS 40, GAS 117 and twoadditional GAS antigens selected from the first antigen group. Preferredcombinations include GAS 40, GAS 117, and two GAS antigens selected fromthe group consisting of GAS 39, GAS 57, GAS 202, GAS 294, GAS 527, GAS533, and GAS 511.

In another embodiment, the combination of GAS antigens consists of GAS40 and three additional GAS antigens selected from the first antigengroup. Preferred combinations include GAS 40 and three additional GASantigens selected from the group consisting of GAS 39, GAS 57, GAS 117,GAS 202, GAS 294, GAS 527, GAS 533, and GAS 511. In one embodiment, thecombination of GAS antigens consists of GAS 117 and three additionalantigens selected from the first antigen group.

The combination of GAS antigens may consist of five GAS antigensselected from the first antigen group. In one embodiment, thecombination of GAS antigens consists of GAS 40, GAS 117 and threeadditional GAS antigens selected from the first antigen group. Preferredcombinations include GAS 40, GAS 117 and three additional GAS antigensselected from the group consisting of GAS 39, GAS 57, GAS 202, GAS 294,GAS 527, GAS 533, and GAS 511. In another embodiment, the combination ofGAS antigens consists of GAS 40 and four additional GAS antigensselected from the first antigen group. Preferred combinations includeGAS 40 and four additional GAS antigens selected from the groupconsisting of GAS 39, GAS 57, GAS 117, GAS 202, GAS 294, GAS 527, GAS533, and GAS 511. In one embodiment, the combination of GAS antigensconsists of GAS 117 and four additional GAS antigens selected from thefirst antigen group.

The combination of GAS antigens may consist of eight GAS antigensselected from the first antigen group. In one embodiment, thecombination of GAS antigens consists of GAS 40, GAS 117 and sixadditional GAS antigens selected from the first antigen group. In oneembodiment, the combination of GAS antigens consists of GAS 40 and sevenadditional GAS antigens selected from the first antigen group. In oneembodiment, the combination of GAS antigens consists of GAS 117 andseven additional GAS antigens selected from the first antigen group.

The combination of GAS antigens may consist of ten GAS antigens selectedfrom the first antigen group. In one embodiment, the combination of GASantigens consists of GAS 40, GAS 117 and eight additional GAS antigensselected from the first antigen group. In one embodiment, thecombination of GAS antigens consists of GAS 40 and nine additional GASantigens selected from the first antigen group. In one embodiment, thecombination of GAS antigens consists of GAS 117 and nine additional GASantigens selected from the first antigen group.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 identifies a leader peptide sequence, two coiled-coil sequences,a leucine zipper sequence and a transmembrane sequence within a GAS 40amino acid sequence (SEQ ID NO:1).

FIG. 2 depicts a schematic of GAS 40 identifying a leader peptidesequence, two coiled-coil sequences, a leucine zipper sequence and atransmembrane sequence, as well as coiled-coil regions of GAS 40 whichhave low level homology with other Streptococcal proteins of known orpredicted function.

FIGS. 3A-3D include the BLAST alignment analysis of identifiedcoiled-oil regions of GAS 40 (SEQ ID NO:1) with other Streptococcusbacteria. FIG. 3A, SpA precursor of S. gordonii (SEQ ID NO:15); FIG. 3B,SpB precursor of S. gordonii (SEQ ID NO:16); FIG. 3C, PspA precursor ofS. pneumoniae (SEQ ID NO:17); FIG. 3D, SpB precursor of S. gordonii (SEQID NO:16).

FIG. 4 provides predicted secondary structure for an amino acid sequenceof GAS 40 (SEQ ID NO:1).

FIG. 5 schematically depicts the location of GAS 40 within the GASgenome. It also includes comparison schematic depicting a GAS mutantwith GAS 40 deleted. Further details on these schematics demonstrate thelikelihood that GAS 40 was acquired by horizontal transfer through atransposon factor.

FIG. 6 provides comparison FACS analysis depicting the surface exposureof GAS 40 in a wild type strain (and no surface exposure in the GAS 40deletion mutant).

FIG. 7 presents opsonophagocytosis data for GAS 40 (in variousexpression constructs).

FIG. 8 presents immunization and challenge data for several GAS antigensof the invention.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the invention provides compositions comprising acombination of GAS antigens, wherein the combinations can be selectedfrom groups of antigens which Applicants have identified as beingparticularly suitable for immunization purposes, particularly when usedin combination. In particular, the invention includes compositionscomprising GAS 40. GAS 40 and the other GAS antigens of the firstantigen group are described in more detail below. Genomic sequences ofat least three GAS strains are publicly available. The genomic sequenceof an M1 GAS strain is reported at Ferretti et al, PNAS (2001)98(8):4658-4663. The genomic sequence of an M3 GAS strain is reported atBeres et al., PNAS (2002) 99(15):10078-10083. The genomic sequence of anM18 GAS strain is reported at Smooet et al., PNAS (2002)99(7):4668-4673. Preferably, the GAS antigens of the invention comprisepolynucleotide or amino acid sequence of an M1, M3 or M18 GAS strains.More preferably, the GAS antigens of the invention comprise apolynucleotide or amino acid sequence of an M1 strain.

As there will be variance among the identified GAS antigens between GASM types and GAS strain isolates, references to the GAS amino acid orpolynucleotide sequences of the invention preferably include amino acidor polynucleotide sequences having sequence identity thereto. Preferredamino acid or polynucleotide sequences have 50% or more sequenceidentity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5% or more). Similarly, references to theGAS amino acid or polynucleotide sequences of the invention preferablyinclude fragments of those sequences, (i.e., fragments which retain orencode for the immunological properties of the GAS antigen). Preferredamino acid fragments include at least n consecutive amino acids, whereinn is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, 100, 150, 200, 250 or more). Preferred polynucleotidefragments include at least n consecutive polynucleotides, wherein n is12 or more (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). In oneembodiment, the amino acid or polynucleotide fragments of the inventionare not identical to amino acid or polynucleotide sequences from other(non-GAS) bacteria (e.g., the fragments are not identical to sequencesin other Streptococcus bacteria).

(1) GAS 40

GAS 40 corresponds to M1 GenBank accession numbers GI: 13621545 and GI:15674449, to M3 GenBank accession number GI: 21909733, to M18 GenBankaccession number GI: 19745402, and is also referred to as ‘Spy0269’(M1), ‘SpyM3_(—)0197’ (M3), ‘SpyM18_(—)0256’ (M18) and ‘prgA’. GAS 40has also been identified as a putative surface exclusion protein. Aminoacid and polynucleotide sequences of GAS 40 from an M1 strain are setforth below and in the sequence listing as SEQ ID NOS: 1 and 2.

SEQ ID NO: 1MDLEQTKPNQVKQKIALTSTIALLSASVGVSHQVKADDRASGETKASNTHDDSLPKPETIQEAKATIDAVEKTLSQQKAELTELATALTKTTAEINHLKEQQDNEQKALTSAQEIYTNTLASSEETLLAQGAEHQRELTATETELHNAQADQHSKETALSEQKASISAETTRAQDLVEQVKTSEQNIAKLNAMISNPDAITKAAQTANDNTKALSSELEKAKADLENQKAKVKKQLTEELAAQKAALAEKEAELSRLKSSAPSTQDSIVGNNTMKAPQGYPLEELKKLEASGYIGSASYNNYYKEHADQIIAKASPGNQLNQYQDIPADRNRFVDPDNLTPEVQNELAQFAAHMINSVRRQLGLPPVTVTAGSQEFARLLSTSYKKTHGNTRPSFVYGQPGVSGHYGVGPHDKTIIEDSAGASGLIRNDDNMYENIGAFNDVHTVNGIKRGIYDSIKYMLFTDHLHGNTYGHAINFLRVDKHNPNAPVYLGFSTSNVGSLNEHFVMFPESNIANHQRFNKTPIKAVGSTKDYAQRVGTVSDTIAAIKGKVSSLENRLSAIHQEADIMAAQAKVSQLQGKLASTLKQSDSLNLQVRQLNDTKGSLRTELLAAKAKQAQLEATRDQSLAKLASLKAALHQTEALAEQAAARVTALVAKKAHLQYLRDFKLNPNRLQVIRERIDNTKQDLAKTTSSLLNAQEALAALQAKQSSLEATIATTEHQLTLLKTLANEKEYRHLDEDIATVPDLQVAPPLTGVKPLSYSKIDTTPLVQEMVKETKQLLEASARLAAENTSLVAEALVGQTSEMVASNAIVSKITSSITQPSSKTSYCSGSSTTSNLISDVDESTQRALKAGVVMLAAVGLTGFRFRKESK SEQ ID NO: 2ATGGACTTAGAACAAACGAAGCCAAACCAAGTTAAGCAGAAAATTGCTTTAACCTCAACAATTGCTTTATTGAGTGCCAGTGTAGGCGTATCTCACCAAGTCAAAGCAGATGATAGAGCCTCAGGAGAAACGAAGGCGAGTAATACTCACGACGATAGTTTACCAAAACCAGAAACAATTCAAGAGGCAAAGGCAACTATTGATGCAGTTGAAAAAACTCTCAGTCAACAAAAAGCAGAACTGACAGAGCTTGCTACCGCTCTGACAAAAACTACTGCTGAAATCAACCACTTAAAAGAGCAGCAAGATAATGAACAAAAAGCTTTAACCTCTGCACAAGAAATTTACACTAATACTCTTGCAAGTAGTGAGGAGACGCTATTAGCCCAAGGAGCCGAACATCAAAGAGAGTTAACAGCTACTGAAACAGAGCTTCATAATGCTCAAGCAGATCAACATTCAAAAGAGACTGCATTGTCAGAACAAAAAGCTAGCATTTCAGCAGAAACTACTCGAGCTCAAGATTTAGTGGAACAAGTCAAAACGTCTGAACAAAATATTGCTAAGCTCAATGCTATGATTAGCAATCCTGATGCTATCACTAAAGCAGCTCAAACGGCTAATGATAATACAAAAGCATTAAGCTCAGAATTGGAGAAGGCTAAAGCTGACTTAGAAAATCAAAAAGCTAAAGTTAAAAAGCAATTGACTGAAGAGTTGGCAGCTCAGAAAGCTGCTCTAGCAGAAAAAGAGGCAGAACTTAGTCGTCTTAAATCCTCAGCTCCGTCTACTCAAGATAGCATTGTGGGTAATAATACCATGAAAGCACCGCAAGGCTATCCTCTTGAAGAACTTAAAAAATTAGAAGCTAGTGGTTATATTGGATCAGCTAGTTACAATAATTATTACAAAGAGCATGCAGATCAAATTATTGCCAAAGCTAGTCCAGGTAATCAATTAAATCAATACCAAGATATTCCAGCAGATCGTAATCGCTTTGTTGATCCCGATAATTTGACACCAGAAGTGCAAAATGAGCTAGCGCAGTTTGCAGCTCACATGATTAATAGTGTAAGAAGACAATTAGGTCTACCACCAGTTACTGTTACAGCAGGATCACAAGAATTTCCAAGATTACTTAGTACCAGCTATAAGAAAACTCATGGTAATACAAGACCATCATTTGTCTACGGACAGCCAGGGGTATCAGGGCATTATGGTGTTGGGCCTCATGATAAAACTATTATTGAAGACTCTGCCGGAGCGTCAGGGCTCATTCGAAATGATGATAACATGTACGAGAATATCGGTGCTTTTAACGATGTGCATACTGTGAATGGTATTAAACGTGGTATTTATGACAGTATCAAGTATATGCTCTTTACAGATCATTTACACGGAAATACATACGGCCATGCTATTAACTTTTTACGTGTAGATAAACATAACCCTAATGCGCCTGTTTACCTTGGATTTTCAACCAGCAATGTAGGATCTTTGAATGAACACTTTGTAATGTTTCCAGAGTCTAACATTGCTAACCATCAACGCTTTAATAAGACCCCTATAAAAGCCGTTGGAAGTACAAAAGATTATGCCCAAAGAGTAGGCACTGTATCTGATACTATTGCAGCGATCAAAGGAAAAGTAAGCTCATTAGAAAATCGTTTGTCGGCTATTCATCAAGAAGCTGATATTATGGCAGCCCAAGCTAAAGTAAGTCAACTTCAAGGTAAATTAGCAAGCACACTTAAGCAGTCAGACAGCTTAAATCTCCAAGTGAGACAATTAAATGATACTAAAGGTTCTTTGAGAACAGAATTACTAGCAGCTAAAGCAAAACAAGCACAACTCGAAGCTACTCGTGATCAATCATTAGCTAAGCTAGCATCGTTGAAAGCCGCACTGCACCAGACAGAAGCCTTAGCAGAGCAAGCCGCAGCCAGAGTGACAGCACTGGTGGCTAAAAAAGCTCATTTGCAATATCTAAGGGACTTTAAATTGAATCCTAACCGCCTTCAAGTGATACGTGAGCGCATTGATAATACTAAGCAAGATTTGGCTAAAACTACCTCATCTTTGTTAAATGCACAAGAAGCTTTAGCAGCCTTACAAGCTAAACAAAGCAGTCTAGAAGCTACTATTGCTACCACAGAACACCAGTTGACTTTGCTTAAAACCTTAGCTAACGAAAAGGAATATCGCCACTTAGACGAAGATATAGCTACTGTGCCTGATTTGCAAGTAGCTCCACCTCTTACGGGCGTAAAACCGCTATCATATAGTAAGATAGATACTACTCCGCTTGTTCAAGAAATGGTTAAAGAAACGAAACAACTATTAGAAGCTTCAGCAAGATTAGCTGCTGAAAATACAAGTCTTGTAGCAGAAGCGCTTGTTGGCCAAACCTCTGAAATGGTAGCAAGTAATGCCATTGTGTCTAAAATCACATCTTCGATTACTCAGCCCTCATCTAAGACATCTTATGGCTCAGGATCTTCTACAACGAGCAATCTCATTTCTGATGTTGATGAAAGTACTCAAAGAGCTCTTAAAGCAGGAGTCGTCATGTTGGCAGCTGTCGGCCTCACAGGATTTAGGTTCCGTAAGGAATCTAAGTGA

Preferred GAS 40 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 1; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 1, wherein n is 7 or more (e.g. 8,10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These GAS 40 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 1.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 1. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 1.

For example, in one embodiment, the underlined amino acid sequence atthe N-terminus (leader sequence) of SEQ ID NO: 1 is removed. (The aminoacid and polynucleotide sequences for this N terminal leader sequenceare listed in the sequence listing as SEQ ID NOS: 3 and 4. The aminoacid and polynucleotide sequences for the remaining GAS 40 fragment arelisted in the sequence listing as SEQ ID NOS: 5 and 6.)

As another example, in one embodiment, the underlined amino acidsequence at the C-terminus (transmembrane region) of SEQ ID NO: 1 isremoved. (The amino acid and polynucleotide sequences for thistransmembrane region are listed in the sequence listing as SEQ ID NOS: 7and 8. The amino acid and polynucleotide sequences for the remaining GAS40 fragment are listed in the sequence listing as SEQ ID NOS: 9 and 10).

Other fragments may omit one or more domains of the protein (e.g.omission of a signal peptide, of a cytoplasmic domain, of atransmembrane domain, or of an extracellular domain).

Further illustration of domains within GAS 40 is shown in FIGS. 1 and 2.As shown in these figures, an amino acid sequence for GAS 40 (SEQ IDNO: 1) contains a leader peptide sequence within amino acids 1-26 (forexample SEQ ID NO: 3), a first coiled-coil region within amino acids58-261 (SEQ ID NO: 12), a second coiled coil region generally withinamino acids 556-733 (SEQ ID NO: 13), a leucine zipper region withinamino acids 673-701 (SEQ ID NO: 14) and a transmembrane region withinamino acids 855-866 (SEQ ID NO: 11). FIG. 1 depicts these regions withinan amino acid sequence for GAS 40, while FIG. 2 depicts these regionsschematically along the length of the GAS 40 protein.

The coiled-coil regions identified within GAS 40 are likely to formalpha helical coils. These structures are frequently involved inoligomerization interactions, for example between different regions ofthe protein or between regions of two separate proteins. The leucinezipper motif within the second coiled-coil region contains a series ofleucine (or isoleucine) amino acid residues, spaced in such a way as toallow the protein to form a specialized oligomerization interactionbetween two alpha helices. In a leucine zipper motif, preferably, thereare six amino acid residues interspaced between the repeating leucineresidues. In a leucine zipper oligomeric structure, the alpha helicesare thought to be held together by hydrophobic interactions betweenleucine residues, which are located on one side of each helix. Leucinezipper motifs are frequently involved in dimerization interactions. Thelocation of the leucine zipper motif within the coiled-coil regionfurther indicates the likelihood that this region of the GAS 40 proteinis involved in an oligomerization interaction.

FIG. 2 also illustrates that there is low level homology between some ofthe identified regions of GAS 40 and other Streptococcal proteins withknown or predicted two dimensional structures or surface localization.Such low level homology may indicate a similar secondary structures oreven function. For example, amino acids 33 to 324 of GAS 40, includingthe first coiled-coil region, has approximately 22% sequence identity toa region (amino acids 112 to 392) of a protein from Streptococcusgordonii called streptococcal surface protein A (“SpA”) precursor(Genbank reference GI 25990270, SEQ ID NO: 15). This protein is thoughtto be a surface protein adhesion, involved in the adhesion of thatStreptococcus with mammalian host cell membranes. The S. gordonii SpA isa member of streptococcal antigen I/II family of protein adhesions andrecognizes salivary agglutinin glycoprotein (gp-340) and type I collagenAmino acids 33 to 258 of GAS 40 also show low level sequence identify(23%) with another S. gordonii protein, Streptococcal surface protein Bprecursor (Genbank reference GI 25055226, SEQ ID NO: 16).

A similar region of GAS 40 which also overlaps with the firstcoiled-coil region (amino acids 43-238) demonstrates about 23% sequenceidentity to a region (amino acids 43-238) of a protein fromStreptococcus pneumoniae called surface protein pspA precursor (Genbankreference GI 282335, SEQ ID NO: 17). The amino terminal domain of pspAis thought to be essential for full pneumococcal virulence, andmonoclonal antibodies raised against it protect mice againstpneumococcal infections. The pspA domain has a monomeric form with anaxial shape ratio of approximately 1:12, typical of fibrous proteins.Sequence analyses indicates an alpha-helical coiled-coil structure forthis monomeric molecule with only few loop-type breaks in helicity.

The second coiled-coil region of GAS 40 has about 46% sequence identityto a region (amino acids 509-717) of a protein from Streptococcus equicalled immunoreactive protein Se89.9 (Genbank reference GI 2330384, SEQID NO: 18) (the full length sequence for Se89.9 is also available athttp://pedant.gsf.de). This Streptococcus equi protein is predicted tobe surface exposed. BLAST alignment of each of these Streptococcalsequences with GAS 40 is presented in FIG. 3.

Further illustration of the two dimensional structure of GAS 40 is shownin FIG. 4. First, FIG. 4( a) presents predicted secondary structureanalysis aligned against the amino acid sequence for GAS 40. Thepredicted alpha helical regions in FIG. 4 generally correspond to thepreviously noted coiled-coil regions. In FIG. 4( b), PairCoil predictionis used to predict the location of putative coiled-coils. Here, two coilregions are identified, generally corresponding to the first and secondcoiled coil regions. FIG. 4( c) highlights the leucine zipper region andillustrates the regularly repeating leucine (or isoleucine) amino acidresidues which are likely to participate in the leucine zipper.

Accordingly, the first coiled-coil region of GAS 40 comprises an aminoacid sequence of at least ten (e.g., at least 10, 13, 15, 18, 20, 25,30, 35, 40, 50, 70, 90, 100 or more) consecutive amino acid residues,selected from the N-terminal half of a full length GAS 40 sequence, andpredicted to form an alpha-helical complex based on the functionalcharacteristics of the amino acid residues in the sequence. SEQ ID NO:12 is a preferred first coiled-coil region of GAS 40.

The second coiled-coil region of GAS 40 comprises an amino acid sequenceof at least ten (e.g., at least 10, 13, 15, 18, 20, 25, 30, 35, 40, 50,70, 90, 100 or more) consecutive amino acid residues, selected from theC-terminal half of a full length GAS 40 sequence, and predicted to forman alpha-helical complex based on the functional characteristics of theamino acid residues in the sequence. The second coiled-coil regionpreferably includes a leucine zipper motif. SEQ ID NO: 13 is a preferredsecond coiled-coil region of GAS 40.

The coiled-coil regions of GAS 40 are likely to be involved in theformation of oligomers such as dimers or trimers. Such oligomers couldbe homomers (containing two or more GAS 40 proteins oligomerizedtogether) or heteromers (containing one or more additional GAS proteinsoligomerized with GAS 40). Alternatively, the first and secondcoiled-coil regions may be interacting together within the GAS 40protein to form oligomeric reactions between the first and secondcoiled-coil regions.

Accordingly, in one embodiment, the compositions of the inventioninclude a GAS 40 antigen in the form of an oligomer. The oligomer maycomprise two more GAS 40 antigens or fragments thereof, or it maycomprise GAS 40 or a fragment thereof oligomerized to a second GASantigen. Preferred GAS 40 fragments comprise an amino acid sequenceselected from the group consisting of the first coiled-coil region andthe second coiled-coil region. Such preferred GAS 40 fragments may beused alone or in the combinations of the invention.

The GAS polynucleotides and amino acid sequences of the invention may bemanipulated to facilitate or optimise recombinant expression. Forexample, the N-terminal leader sequence may be replaced with a sequenceencoding for a tag protein such as polyhistidine (“HIS”) or glutathioneS-transferase (“GST”). Such tag proteins may be used to facilitatepurification, detection and stability of the expressed protein.Variations of such modifications for GAS 40 are discussed below. Suchmodifications can be applied to any of the GAS proteins of theinvention.

An example of a GAS 40 sequence with both a GST and a HIS tag is denotedherein as “GST 40 HIS”. This construct includes a GAS 40 sequence wherethe leader sequence is removed, a GST tag coding sequence is added tothe N-terminus, and a HIS tag coding sequence is added to the C-terminus(using, for example, a pGEXNNH vector with NdeI and NotI restrictionsites). Polynucleotide and amino acid sequences for the fused region ofthe GST tag, the GAS 40 sequence and the C-terminus HIS tag of GST 40HIS are shown in SEQ ID NOS: 19 and 20.

Alternatively, a single tag sequence may be used. An example of a GAS 40sequence with just a HIS tag is denoted as “40a-HIS”. This constructincludes a GAS 40 sequence where the N-terminus leader sequence and theC-terminus containing the transmembrane sequence is removed. In thisconstruct, the HIS tag sequence is added to the C-terminus (using forexample, a cloning vector such as pET21b+(Novagen) at the NdeI and NotIrestriction sites). Polynucleotide and amino acid sequences for 40a-HISare shown in SEQ ID NOS. 21 and 22.

In addition to the addition of purification tags, recombinant expressionmay also be facilitated by optimising coding sequences to those moreabundant or accessible to the recombinant host. For example, thepolynucleotide sequence AGA encodes an arginine amino acid residue.Arginine may also be encoded by the polynucleotide sequence CTG. ThisCTG codon is preferred by the translational enzymes in E. coli. In the40a-HIS polynucleotide sequence SEQ ID NO 21, a C-terminus CTG codingfor arginine has been replaced with CGT.

The following codons are generally underrepresented in E. coli: AGA, AGGand CGA. When these codons occur in a GAS polynucleotide sequence, theymay be replaced with one of the other two optional codons encoding forthe same amino acid residue.

A total of three ATG codons are optimised to CTG in the “40a-RR-HIS”construct, SEQ ID NOS 23 and 24. SEQ ID NO 23 is also shown below, withthe optimised codons underlined. (other than the additional codonoptimisation, 40a-RR-HIS is identical to 40a-HIS.)

SEQ ID N: 23ATGAGTGTAGGCGTATCTCACCAAGTCAAAGCAGATGATAGAGCCTCAGGAGAAACGAAGGCGAGTAATACTCACGACGATAGTTTACCAAAACCAGAAACAATTCAAGAGGCAAAGGCAACTATTGATGCAGTTGAAAAAACTCTCAGTCAACAAAAAGCAGAACTGACAGAGCTTGCTACCGCTCTGACAAAAACTACTGCTGAAATCAACCACTTAAAAGAGCAGCAAGATAATGAACAAAAAGCTTTAACCTCTGCACAAGAAATTTACACTAATACTCTTGCAAGTAGTGAGGAGACGCTATTAGCCCAAGGAGCCGAACATCAAAGAGAGTTAACAGCTACTGAAACAGAGCTTCATAATGCTCAAGCAGATCAACATTCAAAAGAGACTGCATTGTCAGAACAAAAAGCTAGCATTTCAGCAGAAACTACTCGAGCTCAAGATTTAGTGGAACAAGTCAAAACGTCTGAACAAAATATTGCTAAGCTCAATGCTATGATTAGCAATCCTGATGCTATCACTAAAGCAGCTCAAACGGCTAATGATAATACAAAAGCATTAAGCTCAGAATTGGAGAAGGCTAAAGCTGACTTAGAAAATCAAAAAGCTAAAGTTAAAAAGCAATTGACTGAAGAGTTGGCAGCTCAGAAAGCTGCTCTAGCAGAAAAAGAGGCAGAACTTAGTCGTCTTAAATCCTCAGCTCCGTCTACTCAAGATAGCATTGTGGGTAATAATACCATGAAAGCACCGCAAGGCTATCCTCTTGAAGAACTTAAAAAATTAGAAGCTAGTGGTTATATTGGATCAGCTAGTTACAATAATTATTACAAAGAGCATGCAGATCAAATTATTGCCAAAGCTAGTCCAGGTAATCAATTAAATCAATACCAAGATATTCCAGCAGATCGTAATCGCTTTGTTGATCCCGATAATTTGACACCAGAAGTCCAAAATGAGCTAGCGCAGTTTGCAGCTCACATGATTAATAGTGTA cGtcGtCAATTAGGTCTACCACCAGTTACTGTTACAGCAGGATCACAAGAATTTGCAAGATTACTTAGTACCAGCTATAAGAAAACTCATGGTAATACAAGACCATCATTTGTCTACGGACAGCCAGGGGTATCAGGGCATTATGGTGTTGGGCCTCATGATAAAACTATTATTGAAGACTCTGCCGGAGCGTCAGGGCTCATTCGAAATGATGATAACATGTACGAGAATATCGGTGCTTTTAACGATGTGCATACTGTGAATGGTATTAAACGTGGTATTTATGACAGTATCAAGTATATGCTCTTTACAGATCATTTACACGGAAATACATACGGCCATGCTATTAACTTTTTACGTGTAGATAAACATAACCCTAATGCGCCTGTTTACCTTGGATTTTCAACCAGCAATGTAGGATCTTTGAATGAACACTTTGTAATGTTTCCAGAGTCTAACATTGCTAACCATCAACGCTTTAATAAGACCCCTATAAAAGCCGTTGGAAGTACAAAAGATTATGCCCAAAGAGTAGGCACTGTATCTGATACTATTGCAGCGATCAAAGGAAAAGTAAGCTCATTAGAAAATCGTTTGTCGGCTATTCATCAAGAAGCTGATATTATGGCAGCCCAAGCTAAAGTAAGTCAACTTCAAGGTAAATTAGCAAGCACACTTAAGCAGTCAGACAGCTTAAATCTCCAAGTGAGACAATTAAATGATACTAAAGGTTCTTTGAGAACAGAATTACTAGCAGCTAAAGCAAAACAAGCACAACTCGAAGCTACTCGTGATCAATCATTAGCTAAGCTAGCATCGTTGAAAGCCGCACTGCACCAGACAGAAGCCTTAGCAGAGCAAGCCGCAGCCAGAGTGACAGCACTGGTGGCTAAAAAAGCTCATTTGCAATATCTAAGGGACTTTAAATTGAATCCTAACCGCCTTCAAGTGATACGTGAGCGCATTGATAATACTAAGCAAGATTTGGCTAAAACTACCTCATCTTTGTTAAATGCACAAGAAGCTTTAGCAGCCTTACAAGCTAAACAAAGCAGTCTAGAAGCTACTATTGCTACCACAGAACACCAGTTGACTTTGCTTAAAACCTTAGCTAACGAAAAGGAATATCGCCACTTAGACGAAGATATAGCTACTGTGCCTGATTTGCAAGTAGCTCCACCTCTTACGGGCGTAAAACCGCTATCATATAGTAAGATAGATACTACTCCGCTTGTTCAAGAAATGGTTAAAGAAACGAAACAACTATTAGAAGCTTCAGCAAGATTAGCTGCTGAAAATACAAGTCTTGTAGCAGAAGCGCTTGTTGGCCAAACCTCTGAAATGGTAGCAAGTAATGCCATTGTGTCTAAAATCACATCTTCGATTACTCAGCCCTCATCTAAGACATCTTATGGCTCAGGATCTTCTACAACGAGCAATCTCATTTCTGATGTTGATGAAAGTACTCAA cGt GCGGCCGCACTCGAGCACCACCACCACCACCACCAC

Codon optimisation can also be used without a purification tag.Construct “40a-RR-Nat”, SEQ ID NOS: 25 and 26, provides such an example.This construct comprises GAS 40 without the N-terminus leader sequenceand the C-terminus transmembrane sequence, with three codonoptimisations (and does not include a HIS tag sequence).

Different cloning vectors can be used to optimise expression indifferent host cells or under different culture conditions. The abovediscussed constructs used pET21b+(Novagen) vector which includes an IPTGinducible promoter. As an alternative, an E. coli/B. subtilis expressionshuttle vector such as pSM214gNH may be used. This vector uses aconstitutive promoter instead of an IPTG inducible promoter. An exampleof a GAS 40 construct using this vector is denoted as “HIS-40a-NH”, SEQID NOS 27 and 28. In this construct, both the N-terminus leader sequenceand the C-terminus transmembrane sequence are removed, and a HIS tag isadded to the N-terminus. Additional N-terminus amino acids areintroduced with the cloning. In addition, two nucleotide changes whichmost likely occurred during PCR are indicated—neither of these changesresults in amino acid changes.

As another alternative, the pSM214gCH shuttle vector may be used. Anexample of a GAS 40 construct using this vector is denoted as“HIS-40a-CH”, SEQ ID NOS: 29 and 30. In this construct, the N-terminusleader sequence and the C-terminus transmembrane sequence are removedand the HIS tag is placed at the C-terminus Two additional amino acidsare also introduced at the amino terminus. Three nucleotide changesintroduced with the cloning are shown in the DNA sequence, with aresulting amino acid change indicated in the protein sequence (fromamino acid F to S).

Codon optimisation can also be used with these alternative cloningvectors. GAS 40 construct “HIS-40a-RR-NH” comprises the “HIS-40a-NH”construct with three codon optimisations. HIS-40a-RR-NH is set forth inthe sequence listing as SEQ ID NOS: 31 and 32.

Accordingly, the GAS antigens used in the invention may be producedrecombinantly using expression constructs which facilitate theirrecombinant production. Preferred sequence modifications to facilitateexpression may be selected from the group consisting of (1) the additionof a purification tag sequence and (2) codon optimisation.

As discussed above, Applicants have identified GAS 40 as beingparticularly suitable for use in immunogenic compositions, either aloneor in combinations. The use of GAS 40 as a particularly effective GASantigen is supported by its association with virulence, its surfacelocalization, its effectiveness in bacterial opsonophagocytosis assaysand in immunization challenge experiments. In addition, the potentialhorizontal acquisition of this virulence factor indicates that thisantigen may be specific to GAS (relative to other Streptococcalbacteria). Further support for the antigenic properties of GAS 40 alsoincludes the identification of coiled-coil regions within the GAS 40 twodimensional structure, and the low level homology of these regions withsurface proteins of other Streptococcal bacteria, including someadhesion proteins.

Applicants' analysis of the location of GAS 40 within the Streptococcalpyogenes genome indicates that this virulence factor was likely acquiredby GAS during evolution as a result of a horizontal gene transfer. FIG.5A depicts GAS 40 within the GAS genome. It is preceded on the 5′ end bya sequence designated “purine operon repressor” or “purR”. It isfollowed on the 3′ end by two sequences encoding ribosomal proteinsdesignated “ribosomal protein S12”, or “rpsL” and “ribosomal protein S7”or “rpsG”. (Amino acid and polynucleotide sequences for these flankinggenes are publicly available on GenBank. (PurR sequences can be foundfor example under Genbank reference GI: 15674250. RpsL sequences can befound for example under Genbank reference GI: 15674250. RpsG sequencescan be found for example under Genbank reference GI: 15674250. Notably,there are two putative promoter sequences designated at the beginning ofthe rpsL sequence. FIG. 5B depicts a GAS mutant where a large portion ofGAS 40 is deleted. The only portion of the GAS 40 sequence remainingcorresponds to polynucleotides 1-97 of SEQ ID NO: 2. The deletionincluded one of the rpsL promoters, leaving the second, P*, intact. (Thehorizontal arrows underlining the schematic indicate the deletedregion.)

FIG. 5C provides additional detail on the wildtype GAS sequence. Here,direct repeat sequences, designated “DR”, are shown flanking the 5′ and3′ ends of GAS 40. (The corresponding sequences in the GAS 40 deletionmutant are identified in FIG. 5D). These direct repeat sequences areapproximately 8 basepairs. One example of such a basepair direct repeatcomprises SEQ ID NO: 136. Such sequence motifs within a bacterial genomefrequently indicate a horizontal gene transfer. In vivo infectionexperiments show that the GAS 40 deletion mutant is several logs lessvirulent than the wild type strain. (Details of this experiment areprovided in Example 2).

The combination of the presence of the flanking direct repeat sequencesand the virulence associated with GAS 40 strongly suggests that the GAS40 sequence was horizontally acquired by Streptococcus pyogenes duringevolution. Notably, while related purR and rpsL are present in relatedStreptococcal bacteria Streptococcus agalactiae and Streptococcusmutants, neither of these bacteria are known to have a GAS 40 homologue.(FIG. 5E schematically depicts the location of purR, rpsL, and rpsGhomologues within S. agalactiae (Group B Streptococcus) and shows thepercent homology of the GBS homologues with the GAS counterparts.Notably, GBS genomes generally only possess one of the direct repeatsequences—and do not contain a pair of the direct repeat sequencesflanking the GAS 40 sequence.)

The surface location of GAS 40 is illustrated by the FACS diagrampresented in FIG. 6. (Discussion of protocols relating to FACS analysisis presented in Example 1). FIG. 6 includes FACS diagrams for both thewild type GAS (designated DSM 2071, an M23 type of GAS) and the deletionmutant (designated DSM 2071.DELTA.40). The absorbance shift for the wildtype strain indicates that GAS 40 is recognized on the surface of thebacteria by anti-GAS 40 antibodies (and that it is not recognized on thesurface of the deletion mutant).

The surface exposure of GAS 40 is further demonstrated by a bacterialopsonophagocytosis assay illustrated in FIG. 7 and in Example 3. In thisassay, GAS strains are incubated with preimmune and immune sera,polymorphonucleates and complement. (The immune sera is generated bymouse immunization with the indicated GAS protein.) Phagocytosis orgrowth of the bacteria are measured logarithmically. Positive histogrambars represent phagocytosis (or bacterial death). Negative histogrambars represent bacterial growth. As shown in FIG. 7, immune seragenerated by each of the GAS 40 expressed proteins resulted in areduction of bacteria (positive histogram bars).

Immunization challenge studies with GAS 40 are discussed in detail inExample 4. As shown in this example, GAS 40, as produced using variousconstructs, provides substantial protection in adult mice. Notably, mostGAS 40 constructs provide almost as much protection as GAS M protein.(GAS M protein is used for comparison as it is known to be highlyimmunogenic. However, M protein is generally not regarded as a suitableGAS vaccine candidate as it varies widely among GAS strains and hasepitopes with potential cross-reactivity with human tissues.) Inaddition, an N-terminus fragment of GAS 40 also provided significantprotection in this model. The N-terminus fragment comprises about 292amino acids from the N-terminus of GAS 40 overlaps with the firstcoiled-coil region. “40N-HIS” (SEQ ID NOS. 33 and 34) is an example ofthis GAS 40 fragment which comprises the coiled-coil region of GAS 40and a C-terminus HIS tag.

(2) GAS 117

GAS 117 corresponds to M1 GenBank accession numbers GI: 13621679 and GI:15674571, to M3 GenBank accession number GI: 21909852, to M18 GenBankaccession number GI: 19745578, and is also referred to as ‘Spy0448’(M1), ‘SpyM3._(—)0316’ (M3), and ‘SpyM18_(—)0491’ (M18). Examples ofamino acid and polynucleotide sequences of GAS 117 of an M1 strain areset forth in the sequence listing as SEQ ID NOS: 35 and 36.

Preferred GAS 117 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, ormore) to SEQ ED NO: 35; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 35, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ormore). These GAS 117 proteins include variants (e.g. allelic variants,homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 35.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 1. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 35. For example, in one embodiment,the underlined amino acid sequence at the N-terminus of SEQ ID NO: 35(shown below) is removed. (SEQ ID NO: 37 comprises the removedN-terminal amino acid sequence. SEQ ID NO: 38 comprises a fragment ofGAS 117 without the N-terminal amino acid sequence). Other fragmentsomit one or more domains of the protein (e.g. omission of a signalpeptide, of a cytoplasmic domain, of a transmembrane domain, or of anextracellular domain).

SEQ ID NO: 35 MTLKKHYYLLSLLALVTVGAAFNTSQSVSAQVYSNEGYHQHLTDEKSHLQYSKDNAQLQLRNILDGYQNDLGRHYSSYYYYNLRTVMGLSSEQDIE KHYEELKNKLHDMYNHY

(3) GAS 130

GAS 130 corresponds to M1 GenBank accession numbers GI: 13621794 and GI:15674677, to M3 GenBank accession number GI: 21909954, to M18 GenBankaccession number GI: 19745704, and is also referred to as ‘Spy0591’(M1), ‘SpyM3_(—)0418’ (M3), and ‘SpyM18_(—)0660’ (M18). GAS 130 haspotentially been identified as a putative protease. Examples of aminoacid and polynucleotide sequences of GAS 130 of an M1 strain are setforth in the sequence listing as SEQ ID NOS: 39 and 40.

Preferred GAS 130 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 39; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 39, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,or more). These GAS 130 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:39. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 39.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 39. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(4) GAS 277

GAS 277 corresponds to M1 GenBank accession numbers GI: 13622962 and GI:15675742, to M3 GenBank accession number GI: 21911206, to M18 GenBankaccession number GI: 19746852, and is also referred to as ‘Spy1939’(M1), ‘SpyM3_(—)1670’ (M3), and ‘SpyM18_(—)2006’ (M18). Amino acid andpolynucleotide sequences of GAS 277 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 41 and 42.

Preferred GAS 277 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 41; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 41, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, ormore). These GAS 277 proteins include variants (e.g. allelic variants,homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 41.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 41. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 41. For example, in one embodiment,the underlined amino acid sequence at the N-terminus of SEQ ID NO: 41(shown below) is removed. (SEQ ID NO: 43 comprises the underlinedN-terminal amino acid. SEQ ID NO: 44 comprises a fragment of GAS 277with the N-terminal amino acid sequence removed). Other fragments omitone or more domains of the protein (e.g. omission of a signal peptide,of a cytoplasmic domain, of a transmembrane domain, or of anextracellular domain).

SEQ ID NO: 41 MTTMQKTISLLSLALLIGLLGTSGKAISVYAQDQHTDNVIAESTISQVSVEASMRGTEPYIDATVTTDQPVRQPTQATITLKDASDNTINSWVYTMAAQQRRFTAWFDLTGQKSGDYHVTVTVHTQEKAVTGQSGTVHFDQNKARKTPTNMQQKDTSKAMTNSVDVDTKAQTNQSANQEIDSTSNPFRSATNHRSTSLKRSTKNEKLTPTASNSQKNGSNKTKMLVDKEEVKPTSKRGFPWVLLGLVVSLAAGLFIAIQKVSRRK

(5) GAS 236

GAS 236 corresponds to M1 GenBank accession numbers GI: 13622264 and GI:15675106, M3 GenBank accession number GI: 21910321, and to M18 GenBankaccession number GI: 19746075, and is also referred to as ‘Spy1126’(M1), ‘SpyM3_(—)0785’ (M3), and ‘SpyM18_(—)1087’ (M18). Amino acid andpolynucleotide sequences of GAS 236 from an M1 strain are set forth inthe sequence listing as SEQ ID NOS: 45 and 46.

Preferred GAS 236 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 45; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 45, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150or more). These GAS 236 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:45. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 45.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 45. For example, in oneembodiment, the underlined amino acid sequence at the N-terminus of SEQID NO: 45 (shown below) is removed. (SEQ ID NO: 47 comprises theN-terminus amino acid sequence. SEQ ID NO: 48 comprises a fragment ofGAS 236 with the N-terminus sequence removed). Other fragments omit oneor more domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

SEQ ID NO: 45 MTQMNYTGKVKRVAIIANGKYQSKRVASKLFSVFKDDPDFYLSKKNPDIVISIGGDGMLLSAFHMYEKELDKVRFVGIHTGHLGFYTDYRDFEVDKLIDNLRKDKGEQISYPILKVAITLDDGRVVKARALNEATVKRIEKTMVADVIINHVKFESFRGDGISVSTPTGSTAYNKSLGGAVLHPTIEALQLTEISSLNNRVFRTLGSSIIIPKKDKIELVPKRLGIYTISIDNKTYQLKNVTKVEYFIDDEKIHFVSSPSHTSFWERVKDAFIGEIDS

(6) GAS 389

GAS 389 corresponds to M1 GenBank accession numbers GI: 13622996 and GI:15675772, to M3 GenBank accession number GI: 21911237, to M18 GenBankaccession number GI: 19746884, and is also referred to as ‘Spy1981’(M1), ‘SpyM3_(—)1701’ (M3), ‘SpyM18_(—)2045’ (M18) and ‘relA’. GAS 389has also been identified as a (p)ppGpp synthetase. Amino acid andpolynucleotide sequences of GAS 389 from an M1 strain are set forth inthe sequence listing as SEQ ID NOS: 49 and 50.

Preferred GAS 389 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 49; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 49, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These GAS 389 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:49. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 49.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 49. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(7) GAS 504

GAS 504 corresponds to M1 GenBank accession numbers GI: 13622806 and GI:15675600, to M3 GenBank accession number GI: 21911061, to M18 GenBankaccession number GI: 19746708, and is also referred to as ‘Spy1751’(M1), ‘SpyM3_(—)1525’, ‘SpyM18_(—)1823’ (M18) and ‘fabK’. GAS 504 hasalso been identified as a putative trans-2-enoyl-ACP reductase II Aminoacid and polynucleotide sequences of GAS 504 of an M1 strain are setforth below and in the sequence listing as SEQ ID NOS: 51 and 52.

Preferred GAS 504 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 51; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 51, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150or more). These GAS 504 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:51. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 51.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 51. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(8) GAS 509

GAS 509 corresponds to M1 GenBank accession numbers GI: 13622692 and GI:15675496, to M3 GenBank accession number GI: 21910899, to M18 GenBankaccession number GI: 19746544, and is also referred to as ‘Spy1618’(M1), ‘SpyM3_(—)1363’ (M3), ‘SpyM18_(—)1627’ (M18) and ‘cysM’. GAS 509has also been identified as a putative O-acetylserine lyase Amino acidand polynucleotide sequences of GAS 509 of an M1 strain are set forth inthe sequence listing as SEQ ID NOS: 53 and 54.

Preferred GAS 509 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 53; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 53, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, ormore). These GAS 509 proteins include variants (e.g. allelic variants,homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 53.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 53. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 53. For example, in one embodiment,the underlined amino acid sequence at the C-terminus of SEQ ID NO: 53(shown below) is removed. (SEQ ID NO: 55 comprises the C-terminus aminoacid sequence. SEQ ID NO: 56 comprises a fragment of GAS 509 with theC-terminus sequence removed). Other fragments omit one or more domainsof the protein (e.g. omission of a signal peptide, of a cytoplasmicdomain, of a transmembrane domain, or of an extracellular domain).

SEQ ID NO: 53 MTKIYKTITELVGQTPIIKLNRLIPNEAADVYVKLEAFNPGSSVKDRIALSMIEAAEAEGLISPGDVIIEFTSGNTGIGLAWVGAAKGYRVIIVMPETMSLERRQIIQAYGAELVLTPGAEGMKGAIAKAETLAIELGAWMPMQFNNPANPSIHEKTTAQEILEAFKEISLDAFVSGVGTGGTLSGVSHVLKKANPETVIYAVEAEESAVLSGQEPGPHKIQGISAGFIPNTLDTKAYDQIIRVKSKDALETARLTGAKEGFLVGISSGAALYAAIEVAKQLGKGKHVLTILPDNGERYLSTELYDVPVIKTK

(9) GAS 366

GAS 366 corresponds to M1 GenBank accession numbers GI: 13622612, GI:15675424 and GI: 30315979, to M3 GenBank accession number GI: 21910712,to M18 GenBank accession number GI: 19746474, and is also referred to as‘Spy1525’ (M1), ‘SpyM3_(—)1176’ (M3), ‘SpyM18_(—)1542’ (M18) and ‘murD’.GAS 366 has also been identified as aUDP-N-acetylemuramoylalanine-D-glutamate ligase or a D-glutamic acidadding enzyme Amino acid and polynucleotide sequences of GAS 366 of anM1 strain are set forth in the sequence listing as SEQ ID NOS: 57 and58.

Preferred GAS 366 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 57; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 57, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150or more). These GAS 366 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:57. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 57.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 57. For example, in oneembodiment, the underlined amino acid sequence at the N-terminus of SEQID NO: 57 (shown below) is removed. (SEQ ID NO: 59 comprises theN-terminus leader sequence. SEQ ID NO: 60 comprises a fragment of GAS366 where the N-terminus sequence is removed). Other fragments omit oneor more domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

SEQ ID NO: 57 MKVISNFQNKKILILGLAKSGEAAAKLLTKLGALVTVNDSKPFDQNPAAQALLEEGIKVICGSHPVELLDENFEYMVKNPGIPYDNPMVKRALAKEIPILTEVELAYFVSEAPIIGITGSNGKTTTTTMIADVLNAGGQSALLSGNIGYPASKVVQKAIAGDTLVMELSSFQLVGVNAFRPHIAVITNLMPTHLDYHGSFEDYVAAKWMIQAQMTESDYLILNANQEISATLAKTTKATVIPFSTQKVVDGAYLKDGILYFKEQAIIAATDLGVPGSHNIENALATIAVAKLSGIADDIIAQCLSHFGGVKHRLQRVGQIKDITFYNDSKSTNILATQKALSGFDNSRLILIAGGLDRGNEFDDLVPDLLGLKQMIILGESAERMKRAANKAEVSYLEARNVAEATELAFKLAQTGDTILLSRANASWDMYP NFEVRGDEFLATFDCLRGDA

(10) GAS 159

GAS 159 corresponds to M1 GenBank accession numbers GI: 13622244 and GI:15675088, to M3 GenBank accession number GI: 21910303, to M18 GenBankaccession number GI: 19746056, and is also referred to as ‘Spy1105’(M1), ‘SpyM3_(—)0767’ (M3), ‘SpyM18_(—)1067’ (M18) and ‘potD’. GAS 159has also been identified as a putative spermidine/putrescine ABCtransporter (a periplasmic transport protein). Amino acid andpolynucleotide sequences of GAS 159 of an M1 strain are set forth belowand in the sequence listing as SEQ ID NOS: 61 and 62.

SEQ ID NO: 61 MRKLYSFLAGVLGVIVILTSLSFILQKKSGSGSQSDKLVIYNWGDYIDPALLKKFTKETGIEVQYETFDSNEAMYTKIKQGGTTYDIAVPSDYTIDKMIKENLLNKLDKSKLVGMDNIGKEFLGKSFDPQNDYSLPYFWGTVGIVYNDQLVDKAPMHWEDLWRPEYKNSIMLIDGAREMLGVGLTTFGYSVNSKNLEQLQAAERKLQQLTPNVKAIVADEMKGYMIQGDAAIGITFSGEASEMLDSNEHLHYIVPSEGSNLWFDNLVLPKTMKHEKEAYAPLNFINRPENAAQNAAYIGYATPNKKAKALLPDEIKNDPAFYPTDDIIKKLEVYDN LGSRWLGIYNDLYLQFKMYRKSEQ ID NO: 62 ATGCGTAAACTTTATTCCTTTCTAGCAGGAGTTTTGGGTGTTATTGTTATTTTAACAAGTCTTTCTTTCATCTTGCAGAAAAGCAGGGCGGAACCACTTACGACATTGCTGTTCCTAGTGATTACACCATTGATAAAATGATCAAAGAAAACCTACTCAATAAGCTTGATAAGTCAAAATTAGTTGGCATGGATAATATCGGGAAAGAATTTTTAGGGAAAAGCTTTGACCCACAAAACGACTATTCTTTGCCTTATTTCTGGGGAACCGTTGGGATTGTTTATAATGATCAATTAGTTGATAAGGCGCCTATGCACTGGGAAGATCTGTGGCGTCCAGAATATAAAAATAGTATTATGCTGATTGATGGAGCGCGTGAAATGCTAGGGGTTGGTTTAACAACTTTTGGTTATAGTGTGAATTCTAAAAATCTAGAGCAGTTGCAGGCAGCCGAGAGAAAACTGCAGCAGTTGACGCCGAATGTTAAAGCCATTGTAGCAGATGAGATGAAAGGCTACATGATTCAAGGTGACGCTGCTATTGGAATTACCTTTTCTGGTGAAGCCAGTGAGATGTTAGATAGTAACGAACACCTTCACTACATCGTGCCTTCAGAAGGGTCTAACCTTTGGTTTGATAATTTGGTACTACCAAAAACCATGAAACACGAAAAAGAAGCTTATGCTTTTTTGAACTTTATCAATCGTCCTGAAAATGCTGCGCAAAATGCTGCATATATTGGTTATGCGACACCAAATAAAAAAGCCAAGGCCTTACTTCCAGATGAGATAAAAAATGATCCTGCTTTTTATCCAACAGATGACATTATCAAAAAATTGGAAGTTTATGACAATTTAGGGTCAAGATGGTTGGGGATTTATAATGATTTATACCTCCAATTTAAAATGTATC GCAAATAA

Preferred GAS 159 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 61; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 61, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150or more). These GAS 159 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:61. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 61.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 61. For example, in oneembodiment, the underlined amino acid sequence at the N-terminus of SEQID NO: 61 (shown below) is removed. (SEQ ID NO: 63 comprises theN-terminus leader amino acid sequence. SEQ ID NO: 64 comprises afragment of GAS 159 where the N-terminus leader amino acid sequence isremoved). In another example, the underlined amino acid sequence at theC-terminus of SEQ ID NO: 61 is removed. (SEQ ID NO: 65 comprises theC-terminus hydrophobic region. SEQ ID NO: 66 comprises a fragment of GAS159 where the C-terminus hydrophobic region is removed. SEQ ID NO: 67comprises a fragment of GAS 159 where both the N-terminus leadersequence and C-terminus hydrophobic region are removed.) Other fragmentsomit one or more domains of the protein (e.g. omission of a signalpeptide, of a cytoplasmic domain, of a transmembrane domain, or of anextracellular domain).

SEQ ID NO: 61 MRKLYSFLAGVLGVIVILTSLSFILQKKSGSGSQSDKLVIYNWGDYIDPALLKKFTKETGIEVQYETFDSNEAMYTKIKQGGTTYDIAVPSDYTIDKMIKENLLNKLDKSKLVGMDNIGKEFLGKSFDPQNDYSLPYFWGTVGIVYNDQLVDKAPMHWEDLWRPEYKNSIMLIDGAREMLGVGLTTFGYSVNSKNLEQLQAAERKLQQLTPNVKAIVADEMKGYMIQGDAAIGITFSGEASEMLDSNEHLHYIVPSEGSNLWFDNLVLPKTMKHEKEAYAFLNFINRPENAAQNAAYIGYATPNKKAKALLPDEIKNDPAFYPTDDIIKKLEVYDN LGSRWLGIYNDLYLQFKMYRK

(11) GAS 217

GAS 217 corresponds to M1 GenBank accession numbers GI: 13622089 and GI:15674945, to M3 GenBank accession number GI: 21910174, to M18 GenBankaccession number GI: 19745987, and is also referred to as ‘Spy0925’(M1), ‘SpyM3_(—)0638’ (M3), and ‘SpyM18_(—)0982’ (M8). GAS 217 has alsobeen identified as a putative oxidoreductase. Amino acid andpolynucleotide sequences of GAS 217 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 68 and 69.

Preferred GAS 217 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 68; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 68, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, ormore). These GAS 217 proteins include variants (e.g. allelic variants,homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 68.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 68. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 68. Other fragments omit one or moredomains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(12) GAS 309

GAS 309 corresponds to M1 GenBank accession numbers GI: 13621426 and GI:15674341, to M3 GenBank accession number GI: 21909633, to M18 GenBankaccession number GI: 19745363, and is also referred to as ‘Spy0124’(M1), ‘SpyM3_(—)0097’ (M3), ‘SpyM18_(—)0205’ (M18), ‘nra’ and ‘rofA’.GAS 309 has also been identified as a regulatory protein and a negativetranscriptional regulator Amino acid and polynucleotide sequences of GAS309 of an M1 strain are set forth in the sequence listing as SEQ ID NOS:70 and 71.

Preferred GAS 309 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 70; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 70, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, ormore). These GAS 309 proteins include variants (e.g. allelic variants,homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 70.Preferred fragments of (b) comprise an epitope front SEQ ID NO: 70.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 70. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(13) GAS 372

GAS 372 corresponds to M1 GenBank accession numbers GI: 13622698 and GI:15675501, to M3 GenBank accession number GI: 21910905, to M18 GenBankaccession number GI: 19746500 and is also referred to as ‘Spy1625’ (M1),‘SpyM3_(—)1369’ (M3), and ‘SpyM18_(—)1634’ (M18). GAS 372 has also beenidentified as a putative protein kinase or a putative eukaryotic-typeserine/threonine kinase. Amino acid and polynucleotide sequences of GAS372 of an M1 strain are set forth in the sequence listing as SEQ ID NOS:72 and 73.

Preferred GAS 372 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 72; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 72, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These GAS 372 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:72. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 72.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 72. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(14) GAS 039

GAS 039 corresponds to M1 GenBank accession numbers GI: 13621542 and GI:15674446, to M3 GenBank accession number GI: 21909730, to M18 GenBankaccession number GI: 19745398 and is also referred to as ‘Spy0266’ (M1),‘SpyM3_(—)0194’ (M3), and ‘SpyM18_(—)0250’ (M18). Amino acid andpolynucleotide sequences of GAS 039 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 74 and 75.

Preferred GAS 039 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 74; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 74, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,or more). These GAS 039 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:74. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 74.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 74. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(15) GAS 042

GAS 042 corresponds to M1 GenBank accession numbers GI: 13621559 and GI:15674461, to M3 GenBank accession number GI: 21909745, to M18 GenBankaccession number GI: 19745415, and is also referred to as ‘Spy0287’(M1), ‘SpyM3_(—)0209’ (M3), and ‘SpyM18_(—)0275’ (M18). Amino acid andpolynucleotide sequences of GAS 042 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 76 and 77.

Preferred GAS 042 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 76; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 76, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,or more). These GAS 042 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:76. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 76.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 76. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(16) GAS 058

GAS 058 corresponds to M1 GenBank accession numbers GI: 13621663 and GI:15674556, to M3 GenBank accession number GI: 21909841, to M18 GenBankaccession number GI: 19745567 and is also referred to as ‘Spy0430’ (M1),‘SpyM3_(—)0305’ (M3), and ‘SpyM18_(—)0477’ (M18). Amino acid andpolynucleotide sequences of GAS 058 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 78 and 79.

Preferred GAS 058 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 98%, 99%, 99.5% or more) to SEQ ID NO: 78;and/or (b) which is a fragment of at least it consecutive amino acids ofSEQ ID NO: 78, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, or more). These GAS 058proteins include variants (e.g. allelic variants, homologs, orthologs,paralogs, mutants, etc.) of SEQ ID NO: 78. Preferred fragments of (b)comprise an epitope from SEQ ID NO: 78. Other preferred fragments lackone or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQID NO: 78. For example, in one embodiment, the underlined amino acidsequence at the N-terminus of SEQ ID NO: 78 (shown below) is removed.(SEQ ID NO: 80 comprises the N-terminal leader sequence. SEQ ID NO: 81comprises a fragment of GAS 58 where the N-terminal leader sequence isremoved.) Other fragments omit one or more domains of the protein (e.g.omission of a signal peptide, of a cytoplasmic domain, of atransmembrane domain, or of an extracellular domain).

SEQ ID NO: 78 MKWSGFMKTKSKRFLNLATLCLALLGTTLLMAHPVQAEVISKRDYMTRFGLGDLEDDSANYPSNLEARYKGYLEGYEKGLKGDDIPERPKIQVPEDVQPSDHGDYRDGYEEGFGEGQHKRDPLETEAEDDSQGGRQEGRQGHQEGADSSDLNVEESDGLSVIDEVVGVIYQAFSTIWTYLSGLF

(17) GAS 290

GAS 290 corresponds to M1 GenBank accession numbers GI: 13622978 and GI:15675757, to M3 GenBank accession number GI: 21911221, to M18 GenBankaccession number GI: 19746869 and is also referred to as ‘Spy1959’ (M1),‘SpyM3_(—)1685’ (M3), and ‘SpyM18_(—)2026’ (M18). Amino acid andpolynucleotide sequences of GAS 290 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 82 and 83.

Preferred GAS 290 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 82; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 82, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ormore). These GAS 290 proteins include variants (e.g. allelic variants,homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 82.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 82. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 82. Other fragments omit one or moredomains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(18) GAS 511

GAS 511 corresponds to M1 GenBank accession numbers GI: 13622798 and GI:15675592, to M3 GenBank accession number GI: 21911053, to M18 GenBankaccession number GI: 19746700 and is also referred to as ‘Spy1743’ (M1),‘SpyM3_(—)1517’ (M3), ‘SpyM18_(—)1815’ (M18) ‘accA’ Amino acid andpolynucleotide sequences of GAS 511 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 84 and 85.

Preferred GAS 511 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 84; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 84, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ormore). These GAS 511 proteins include variants (e.g. allelic variants,homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 84.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 84. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 84. Other fragments omit one or moredomains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(19) GAS 533

GAS 533 corresponds to M1 GenBank accession numbers GI: 13622912 and GI:15675696, to M3 GenBank accession number GI: 21911157, to M18 GenBankaccession number GI: 19746804 and is also referred to as ‘Spy1877’ (M1),‘SpyM3_(—)1621’ (M3), ‘SpyM18_(—)1942’ (M18) and ‘glnA’. GAS 533 has abeen identified as a putative glutamine synthetase. Amino acid andpolynucleotide sequences of GAS 533 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 86 and 87.

Preferred GAS 533 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 86; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 86, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 533 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:86. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 86.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 86. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(20) GAS 527

GAS 527 corresponds to M1 GenBank accession numbers GI: 13622332, GI:15675169, and GI: 24211764, to M3 GenBank accession number GI: 21910381,to M18 GenBank accession number GI: 19746136, and is also referred to as‘Spy1204’ (M1), ‘SpyM3_(—)0845’ (M3), ‘SpyM18_(—)1155’ (M18) and ‘guaA’.GAS 527 has also been identified as a putative GMP synthetase (glutamatehydrolyzing) (glutamate amidotransferase) Amino acid and polynucleotidesequences of GAS 527 of an M1 strain are set forth in the sequencelisting as SEQ ID NOS: 88 and 89.

Preferred GAS 527 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 98%, 99%, 99.5% or more) to SEQ ID NO: 88;and/or (b) which is a fragment of at least n consecutive amino acids ofSEQ ID NO: 88, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). These GAS527 proteins include variants (e.g. allelic variants, homologs,orthologs, paralogs, mutants, etc.) of SEQ ID NO: 88. Preferredfragments of (b) comprise an epitope from SEQ ID NO: 88. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or one or more aminoacids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from theN-terminus of SEQ ID NO: 88. Other fragments omit one or more domains ofthe protein (e.g. omission of a signal peptide, of a cytoplasmic domain,of a transmembrane domain, or of an extracellular domain).

(21) GAS 294

GAS 294 corresponds to M1 GenBank accession numbers GI: 13622306, GI:15675145, and GI: 26006773, to M3 GenBank accession number GI: 21910357,to M18 GenBank accession number GI: 19746111 and is also referred to as‘Spy1173’ (M1), ‘SpyM3_(—)0821’ (M3), ‘SpyM18_(—)1125’ (M18) and ‘gid’.GAS 294 has also been identified as a putative glucose-inhibiteddivision protein. Amino acid and polynucleotide sequences of GAS 294 ofan M1 strain are set forth in the sequence listing as SEQ ID NOS: 90 and91.

Preferred GAS 294 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 90; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 90, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 294 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:90. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 90.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 90. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(22) GAS 253

GAS 253 corresponds to M1 GenBank accession numbers GI: 13622611, GI:15675423, and GI: 21362716, to M3 GenBank accession number GI: 21910711,to M18 GenBank accession number GI: 19746473 and is also referred to as‘Spy1524’ (M1), ‘SpyM3_(—)1175’ (M3), ‘SpyM18_(—)1541’ (M18) and ‘murG’.GAS 253 has also been identified as a putativeundecaprenyl-PP-MurNAc-pentapeptide-UDPG1cNAc GlcNAc transferase Aminoacid and polynucleotide sequences of GAS 253 of an M1 strain are setforth in the sequence listing as SEQ ID NOS: 92 and 93.

Preferred GAS 253 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 92; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 92, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 253 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:92. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 92.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 92. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(23) GAS 529

GAS 529 corresponds to M1 GenBank accession numbers GI: 13622403, GI:15675233, and GI: 21759132, to M3 GenBank accession number GI: 21910446,to M18 GenBank accession number GI: 19746203 and is also referred to as‘Spy1280’ (M1), ‘SpyM3_(—)0910’ (M3), ‘SpyM18_(—)1228’ (M18) and ‘glmS’.GAS 529 has also been identified as a putativeL-glutamine-D-fructose-6-phosphate aminotransferase(Glucosamine-6-phosphate synthase) Amino acid and polynucleotidesequences of GAS 529 of an M1 strain are set forth below and in thesequence listing as SEQ ID NOS: 94 and 95.

Preferred GAS 529 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 94; and/or (b) which is a fragment of at least itconsecutive amino acids of SEQ ID NO: 94, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 529 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:94. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 94.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 94. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(24) GAS 045

GAS 045 corresponds to M3 GenBank accession number GI: 21909751, M18GenBank accession number GI: 19745421 and is referred to as‘SpyM3_(—)0215’ (M3), ‘SpyM18_oppA’ (M18) and ‘oppA’. GAS 045 has beenidentified as an oligopeptide permease Amino acid and polynucleotidesequences of GAS 045 from an M1 strain are set forth in the sequencelisting as SEQ ID NOS: 96 and 97.

Preferred GAS 045 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 96; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 96, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 045 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:96. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 96.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 96. For example, in oneembodiment, the underlined amino acid sequence at the N-terminus of SEQID NO: 96 (shown below) is removed. (SEQ ID NO: 98 comprises theunderlined N-terminal leader sequence. SEQ ID NO: 99 comprises afragment of GAS 45 where the N-terminal leader sequence is removed).Other fragments omit one or more domains of the protein (e.g. omissionof a signal peptide, of a cytoplasmic domain, of a transmembrane domain,or of an extracellular domain).

SEQ ID NO: 96 VTFMKKSKWLAAVSVAILSVSALAACGNKNASGGSEATKTYKYVFVNDPKSLDYILTNGGGTTDVITQMVDGLLENDEYGNLVPSLAKDWKVSKDGLTYTYTLRDGVSWYTADGEEYAPVTAEDFVTGLKHAVDDKSDALYVVEDSIKNLKAYQNGEVDFKEVGVKALDDKTVQYTLNKPESYWNSKTTYSVLFPVNAKFLKSKGKDFGTTDPSSILVNGAYFLSAFTSKSSMEFHKNENYWDAKNVGIESVKLTYSDGSDPGSFYKNFDKGEFSVARLYPNDPTYKSAKKNYADNITYGMLTGDIRHLTWNLNRTSFKNTKKDPAQQDAGKKALNNKDFRQAIQFAFDRASFQAQTAGQDAKTKALRNMLVPPTFVTIGESDFGSEVEKEMAKLGDEWKDVNLADAQDGFYNPEKAKAEFAKAKEALTAEGVTFPVQLDYPVDQANAATVQEAQSFKQSVEASLGKENVIVNVLETETSTHEAQGFYAETPEQQDYDIISSWWGPDYQDPRTYLDIMSPVGGGSVIQKLGIKAGQNKDVVAAAGLDTYQTLLDEAAAITDDNDARYKAYAKAQAYLTDNAVDIPVVALGGTPRVTKAVPFSGGFSWAGSKGPLAYKGMKLQDKPVTVKQYEKAKEKWMKAKAKSNAKYAEKLADHVEK

(25) GAS 095

GAS 095 corresponds to M1 GenBank accession numbers GI: 13622787 and GI:15675582, to M3 GenBank accession number GI: 21911042, to M18 GenBankaccession number GI: 19746634 and is also referred to as ‘Spy 1733’(M1), ‘SpyM3_(—)1506’ (M3), ‘SpyM18_(—)1741’ (M18). GAS 095 has alsoidentified as a putative transcription regulator. Amino acid andpolynucleotide sequences of GAS 095 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 100 and 101.

Preferred GAS 095 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 100; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 100, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 095 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:100. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 100.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 100. For example, in oneembodiment, the underlined amino acid sequence at the N-terminus of SEQID NO: 100 (shown below) is removed. (SEQ ID NO: 102 comprises the aminoacid sequence of the underlined N-terminal leader sequence. SEQ ID NO:103 comprises a fragment of GAS 95 where the N-terminal leader sequenceis removed.) Other fragments omit one or more domains of the protein(e.g. omission of a signal peptide, of a cytoplasmic domain, of atransmembrane domain, or of an extracellular domain).

SEQ ID NO: 100 MKIGKKIVLMFTAIVLTTVLALGVYLTSAYTFSTGELSKTFKDFSTSSNKSDAIKQTRAFSILLMGVDTGSSERASKWEGNSDSMILVTVNPKTKKTTMTSLERDTLTTLSGPKNNENNGVEAKLNAAYAAGGAQMAIMTVQDLLNITIDNYVQINMQGLIDLVNAVGGITVTNEFDFPISIAENEPEYQATVAPGTHKINGEQALVYARMRYDDPEGDYGRQKRQREVIQKVLKKILALDSISSYRKILSAVSSNMQTNIEISSRTIPSLLGYRDALRTIKTYQLKGEDATLSDGGSYQIVTSNHLLEIQNRIRTELGLHKVNQLKTNATVYENLYGSTKSQTVNNNYDSSGQAPSYSDSHSSYANYSSGVDTGQSASTDQDSTASSHRPATPSSSSDALAADESSSSGSGSLVPPANINPQT

(26) GAS 193

GAS 193 corresponds to M1 GenBank accession numbers GI: 13623029 and GI:15675802, to M3 GenBank accession number GI: 21911267, to M18 GenBankaccession number GI: 19746914 and is also referred to as ‘Spy2025’ (M1),‘SpyM3_(—)1731’ (M3), ‘SpyM18_(—)2082’ (M18) and ‘isp’. GAS 193 has alsobeen identified as an immunogenic secreted protein precursor. Amino acidand polynucleotide sequences of GAS 193 of an M1 strain are set forth inthe sequence listing as SEQ ID NOS: 104 and 105.

Preferred GAS 193 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 104; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 104, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 193 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:104. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 104.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 104. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(27) GAS 137

GAS 137 corresponds to M1 GenBank accession numbers GI: 13621842, GI:15674720 and GI: 30173478, to M3 GenBank accession number GI: 21909998,to M18 GenBank accession number GI: 19745749 and is also referred to as‘Spy0652’ (M1), ‘SpyM3_(—)0462’, and ‘SpyM18_(—)0713’ (M18) Amino acidand polynucleotide sequences of GAS 137 of an M1 strain are set forth inthe sequence listing as SEQ ID NOS: 106 and 107.

Preferred GAS 137 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 106; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 106, wherein it is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 137 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:106. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 106.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 106. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(28) GAS 084

GAS 084 corresponds to M1 GenBank accession numbers GI: 13622398 and GI:15675229, to M3 GenBank accession number GI: 21910442, to M18 GenBankaccession number GI: 19746199 and is also referred to as ‘Spy1274’ (M1),‘SpyM3_(—)0906’ and ‘SpyM18_(—)1223’ (M18). GAS 084 has also beenidentified as a putative amino acid ABC transporter/periplasmic aminoacid binding protein. Amino acid and polynucleotide sequences of GAS 084of an M1 strain are set forth in the sequence listing as SEQ ID NOS: 108and 109.

Preferred GAS 084 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 108; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 108, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 084 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:108. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 108.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 108. For example, in oneembodiment, the underlined amino acid sequence at the N-terminus of SEQID NO: 108 (shown below) is removed. (SEQ ID NO: 110 comprises an aminoacid sequence comprising the underlined N-terminal leader sequence ofGAS 84. SEQ ID NO: 111 comprises a fragment of GAS 84 where theN-terminal leader sequence is removed). Other fragments omit one or moredomains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

SEQ ID NO: 108 MIIKKRTVAILAIASSFFLVACQATKSLKSGDAWGVYQKQKSITVGFDNTFVPMGYKDESGRCKGFDIDLAKEVFHQYGLKVNFQAINWDMKEAELNNGKIDVIWNGYSITKERQDKVAFTDSYMRNEQIIVVKKRSDIKTISDMKHKVLGAQSASSGYDSLLRTPKLLKDFIKNKDANQYETFTQAFIDLKSDRIDGILIDKVYANYYLAKEGQLENYRMIPTTFENEAFSVGLRKEDKTLQAKINRAFRVLYQNGKFQAISEKWFGDDVATANIKS

(29) GAS 384

GAS 384 corresponds to M1 GenBank accession numbers GI: 13622908 and GI:15675693, to M3 GenBank accession number GI: 21911154, to M18 GenBankaccession number GI: 19746801 and is also referred to as ‘Spy1874’ (M1),‘SpyM3_(—)1618’ (M3), and ‘SpyM18_(—)1939’ (M18). GAS 384 has also beenidentified as a putative glycoprotein endopeptidase. Amino acid andpolynucleotide sequences of GAS 384 of an M1 strain are set forth in thesequence listing as SEQ ID NOS: 112 and 113.

Preferred GAS 384 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 112; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 112, wherein it is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 384 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:112. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 112.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 112. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(30) GAS 202

GAS 202 corresponds to M1 GenBank accession numbers GI: 13622431 and GI:15675258, to M3 GenBank accession number GI: 21910527, to M18 GenBankaccession number GI: 19746290 and is also referred to as ‘Spy1309’ (M1),‘SpyM3_(—)0991’ (M3), ‘SpyM18_(—)1321’ (M18) and ‘dItD’. GAS 202 hasalso been identified as a putative extramembranal protein. Amino acidand polynucleotide sequences of GAS 202 of an M1 strain are set forth inthe sequence listing as SEQ ID NOS: 114 and 115.

Preferred GAS 202 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 114; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 114, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 202 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:114. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 114.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 114. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(31) GAS 057

GAS 057 corresponds to M1 GenBank accession numbers GI: 13621655 and GI:15674549, to M3 GenBank accession number GI: 21909834, to M18 GenBankaccession number GI: 19745560 and is also referred to as ‘Spy0416’ (M1),‘SpyM3_(—)0298’ (M3), ‘SpyM18_(—)0464’ (M18) and ‘prtS’. GAS 057 hasalso been identified as a putative cell envelope proteinase Amino acidand polynucleotide sequences of GAS 057 of an M1 strain are set forth inthe sequence listing as SEQ ID NOS: 116 and 117.

Preferred GAS 057 proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 116; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 116, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These GAS 057 proteins include variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO:116. Preferred fragments of (1)) comprise an epitope from SEQ ID NO:116. Other preferred fragments lack one or more amino acids (e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/orone or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25or more) from the N-terminus of SEQ ID NO: 116. For example, in oneembodiment, the underlined amino acid sequence at the N-terminus of SEQID NO: 116 (shown below) is removed. (SEQ ID NO: 118 comprises theunderlined N-terminal leader sequence. SEQ ID NO: 119 comprises afragment of GAS 57 where the N-terminal leader sequence is removed.) Inanother example, the underlined amino acid sequence at the C-terminus ofSEQ ID NO: 116 is removed. (SEQ ID NO: 120 comprises the underlinedC-terminal hydrophobic region. SEQ ID NO: 121 comprises a fragment ofGAS 57 where the C-terminal hydrophobic region is removed. SEQ ID NO:122 comprises a fragment of GAS 57 where both the N-terminal leadersequence and the C-terminal hydrophobic region are removed.) Otherfragments omit one or more domains of the protein (e.g. omission of asignal peptide, of a cytoplasmic domain, of a transmembrane domain, orof an extracellular domain).

SEQ ID NO: 116MEKKQRFSLRKYKSGTFSVLIGSVFLVMTTTVAADELSTMSEPTITNHAQQQAQHLTNTELSSAESKSQDTSQITLKTNREKEQSQDLVSEPTTTELADTDAASMANTGSDATQKSASLPPVNTDVHDWVKTKGAWDKGYKGQGKVVAVIDTGIDPAHQSMRISDVSTAKVKSKEDMLARQKAAGINYGSWINDKVVFAHNYVENSDNIKENQFEDFDEDWENFEFDAEAEPKAIKKHKIYRPQSTQAPKETVIKTEETDGSHDIDWTQTDDDTKYESHGMHVTGIVAGNSKEAAATGARFLGIAPEAQVMFMRVFANDIMGSAESLFIKAIEDAVALGADVINLSLGTANGAQLSGSKPLMEAIEKAKKAGVSVVVAAGNERVYGSDHDDPLATNPDYGLVGSPSTGRTPTSVAAINSKWVIQRLMTVKELENRADLNHGKAIYSESVDFKDIKDSLGYDKSHQFAYVKESTDAGYNAQDVKGKTALIERDPNKTYDEMIALAKKHGALGVLIFNNKPGQSNRSMRLTANGMGIPSAFISHEFGKAMSQLNGNGTGSLEFDSVVSKAPSQKGNEMNHFSNWGLTSDGYLKPDITAPGGDIYSTYNDNHYGSQTGTSMASPQIAGASLLVKQYLEKTQPNLPKEKIADIVKNLLMSNAQIHVNPETKTTTSPRQQGAGLLNIDGAVTSGLYVTGKDNYGSISLGNITDTMTFDVTVHNLSNKDKTLRYDTELLTDHVDPQKGRFTLTSHSLKTYQGGEVTVPANGKVTVRVTMDVSQFTKELTKQMPNGYYLEGFVRFRDSQDDQLNRVNIPFVGFKGQFENLAVAEESIYRLKSQGKTGFYFDESGPKDDIYVGKHFTGLVTLGSETNVSTKTISDNGLHTLGTFKNADGKFILEKNAQGNPVLAISPNGDNNQDFAAFKGVFLRKYQGLKASVYHASDKEHKNPLWVSPESFKGDKNFNSDIRFAKSTTLLGTAFSGKSLTGAELPDGHYHYVVSYYPDVVGAKRQEMTFDMILDRQKPVLSQATFDPETNRFKPEPLKDRGLAGVRKDSVFLYERKDNKPYTVTINDSYKYVSVEDNKTFVERQADGSFILPLDKAKLGDFYYMVEDFAGNVAIAKLGDHLPQTLGKTPIKLKLTDGNYQTKETLKDNLEMTQSDTGLVTNQAQLAVVHRNQPQSQLTKMNQDFFISPNEDGNKDFVAFKGLKNNVYNDLTVNVYAKDDHQKQTPIWSSQAGASVSAIESTAWYGITARGSKVMPGDYQYVVTYRDEHGKEHQKQYTISVNDKKPMITQGRFDTINGVDHFTPDKTKALDSSGIVREEVFYLAKKNGRKFDVTEGKDGITVSDNKVYIPKNPDGSYTISKRDGVTLSDYYYLVEDRAGNVSFATLRDLKAVGKDKAVVNFGLDLPVPEDKQIVNFTYLVRDADGKPIENLEYYNNSGNSLILPYGKYTVELLTYDTNAAKLESDKIVSFTLSADNNFQQVTFKITMLATSQITAHFDHLLPEGSRVSLKTAQDQLIPLEQSLYVPKAYGKTVQEGTYEVVVSLPKGYRIEGNTKVNTLPNEVHELSLRLVKVGDASDSTGDHKVMSKNNSQALTASATPTKSTTSATAKALPSTGEKMGLKLRIVGLVLLGLTCVFSRKKSTKD

Representative examples of immunization with GAS antigens of theinvention in the murine mouse model discussed above are summarized inFIG. 8. The first column identifies the GAS antigen used in theexperiment. In some instances purification aspects are referenced inthis list. Also, modifications to the polynucleotide sequence which havebeen made to facilitate the recombinant expression of the antigen aredenoted in the chart with the following annotations: “a”, indicates thatN or C terminal hydrophobic regions have been removed; RR indicatescodon optimisation; “NH” and “CH” correspond to the expression vectorssimilar to those indicated in the GAS 40 construct examples. Where a pvalue is given, it was calculated based on the control HIS stop valuesat the bottom of the chart.

Mice immunized with GAS 40 yielded substantially improved survival rateson challenge—in a collection of over 100 mice immunizations,immunization with GAS 40 yielded over 50% survival. The other GASantigens in the chart offered an amount of protection that, for exampleif combined with GAS 40, could offer improved protection.

The immunogenicity of other known GAS antigens may be improved bycombination with two or more GAS the first antigen group. Such otherknown GAS antigens include a second antigen group consisting of (1) oneor more variants of the M surface protein or fragments thereof, (2)fibronectin-binding protein, (3) streptococcal heme-associated protein,or (4) SagA. These antigens are referred to herein as the “secondantigen group”.

The invention thus includes an immunogenic composition comprising acombination of GAS antigens, said combination consisting of two tothirty-one GAS antigens of the first antigen group and one, two, three,or four GAS antigens of the second antigen group. Preferably, thecombination consists of three, four, five, six, seven, eight, nine, orten GAS antigens from the first antigen group. Still more preferably,the combination consists of three, four or five GAS antigens from thefirst antigen group. Preferably, the combination of GAS antigensincludes either or both of GAS 40 and GAS 117. Preferably, thecombination of GAS antigens includes one or more variants of the Msurface protein.

Each of the GAS antigens of the second antigen group are described inmore detail below.

(1) M Surface Protein

The M protein is a GAS virulence factor which has been associated withboth colonization and resistance to phagocytosis. Over 100 differenttype variants of the M protein have been identified on the basis ofantigenic specificity and M protein is thought to be the major cause ofantigenic shift and antigenic drift in GAS. The M protein also bindsfibrinogen from serum and blocks the binding of complement to theunderlying peptidoglycan. This action is thought to increase GASsurvival within a mammalian host by inhibiting phagocytosis.

Unfortunately, the GAS M protein contains some epitopes which mimicthose of mammalian muscle and connective tissue. Certain GAS M proteinsmay be rheumatogenic since they contain epitopes related to heartmuscle, and may lead to autoimmune rheumatic carditis (rheumatic fever)following an acute infection.

Epitopes having increased bactericidal activity and having decreasedlikelihood of cross-reacting with human tissues have been identified inthe amino terminal region and combined into fusion proteins containingapproximately six, seven, or eight M protein fragments linked in tandem.See Hu et al., Infection & Immunity (2002) 70(4):2171-2177; Dale,Vaccine (1999) 17:193-200; Dale et al., Vaccine 14(10):944-948; WO02/094851 and WO 94/06465. (Each of the M protein variants, fragmentsand fusion proteins described in these references are specificallyincorporated herein by reference.)

Accordingly, the compositions of the invention may further comprise aGAS M surface protein or a fragment or derivative thereof. One or moreGAS M surface protein fragments may be combined together in a fusionprotein. Alternatively, one or more GAS M surface protein fragments arecombined with a GAS antigen or fragment thereof of the first antigengroup. One example of a GAS M protein is set forth in the sequencelisting as SEQ ID NO: 123.

Preferred GAS M proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to a known M protein such as SEQ ID NO: 123; and/or (b) which is afragment of at least n consecutive amino acids of a known M protein suchas SEQ ID NO: 123, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more). These GAS Mproteins include variants (e.g. allelic variants, homologs, orthologs,paralogs, mutants, etc.) of SEQ ID NO: 123. Preferred fragments of (b)comprise an epitope from a known M protein, such as SEQ ID NO: 123.Preferably, the fragment is one of those described in the referencesabove. Preferably, the fragment is constructed in a fusion protein withone or more additional M protein fragments. Other preferred fragmentslack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or more) from the C-terminus and/or one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminusof a known M protein such as SEQ ID NO: 123. Other fragments omit one ormore domains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(2) Fibronectin-Binding Protein

GAS fibronectin-binding protein (‘SfbI’) is a multifunctional bacterialprotein thought to mediate attachment of the bacteria to host cells,facilitate bacterial internalization into cells and to bind to the Fcfragment of human IgG, thus interfering with Fc-receptor mediatedphagocytosis and antibody-dependent cell cytotoxicity Immunization ofmice with SfbI and an ‘H12 fragment’ (encoded by positions 1240-1854 ofthe SfbI gene) are discussed in Schulze et al., Vaccine (2003)21:1958-1964; Schulze et al., Infection and Immunity (2001)69(1):622-625 and Guzman et al., Journal of Infectious Diseases (1999)179:901-906. One example of an amino acid sequence for GAS SfbI is shownin the sequence listing as SEQ ID NO: 124.

Preferred SfbI proteins for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 124; and/or (b) which is a fragment of at least nconsecutive amino acids of SEQ ID NO: 124, wherein n is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, ormore). These SfbI proteins include variants (e.g. allelic variants,homologs, orthologs, paralogs, mutants, etc.) of SEQ ID NO: 124.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 124.Preferably, the fragment is one of those described in the referencesabove. Other preferred fragments lack one or more amino acids (e.g. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminusand/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of SEQ ID NO: 124. Other fragmentsomit one or more domains of the protein (e.g. omission of a signalpeptide, of a cytoplasmic domain, of a transmembrane domain, or of anextracellular domain).

(3) Streptococcal Heme-Associated Protein

The GAS streptococcal heme-associated protein (‘Shp’) has beenidentified as a GAS cell surface protein. It is thought to becotranscribed with genes encoding homologues of an ABC transporterinvolved in iron uptake in gram-negative bacteria. The Shp protein isfurther described in Lei et al., “Identification and Characterization ofa Novel Heme-Associated Cell Surface Protein Made by Streptococcuspyogenes”, Infection and Immunity (2002) 70(8):4494-4500. One example ofa Shp protein is shown in the sequence listing as SEQ ID NO: 125.

Preferred Shp proteins for use with the invention comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 125; and/or (b) which is a fragment of at least n consecutiveamino acids of SEQ ID NO: 125, wherein n is 7 or more (e.g. 8, 10, 12,14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). TheseShp proteins include variants (e.g. allelic variants, homologs,orthologs, paralogs, mutants, etc.) of SEQ ID NO: 125. Preferredfragments of (b) comprise an epitope from SEQ ID NO: 125. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 125. Other fragments omit one or moredomains of the protein (e.g. omission of a signal peptide, of acytoplasmic domain, of a transmembrane domain, or of an extracellulardomain).

(4) SagA

Streptolysin S (SLS), also known as ‘SagA’, is thought to be produced byalmost all GAS colonies. This cytolytic toxin is responsible for thebeta-hemolysis surrounding colonies of GAS grown on blood agar and isthought to be associated with virulence. While the full SagA peptide hasnot been shown to be immunogenic, a fragment of amino acids 10-30 (SagA10-30) has been used to produce neutralizing antibodies. See Dale etal., “Antibodies against a Synthetic Peptide of SagA Neutralize theCytolytic Activity of Streptolysin S from Group A Streptococci”,Infection and Immunity (2002) 70(4):2166-2170. The amino acid sequenceof SagA 10-30 is shown in the sequence listing as SEQ ID NO: 126.

Preferred SagA 10-30 proteins for use with the invention comprise anamino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5% or more) to SEQ ID NO: 126; and/or (b) which is a fragment of atleast n consecutive amino acids of SEQ ID NO: 126, wherein it is 7 ormore (e.g. 8, 10, 12, 14, 16, 18, or 20). These SagA 10-30 proteinsinclude variants (e.g. allelic variants, homologs, orthologs, paralogs,mutants, etc.) of SEQ ID NO: 126.

There is an upper limit to the number of GAS antigens which will be inthe compositions of the invention. Preferably, the number of GASantigens in a composition of the invention is less than 20, less than19, less than 18, less than 17, less than 16, less than 15, less than14, less than 13, less than 11, less than 10, less than 9, less than 8,less than 7, less than 6, less than 5, less than 4, or less than 3.Still more preferably, the number of GAS antigens in a composition ofthe invention is less than 6, less than 5, or less than 4. Still morepreferably, the number of GAS antigens in a composition of the inventionis 3. The GAS antigens used in the invention are preferably isolated,i.e., separate and discrete, from the whole organism with which themolecule is found in nature or, when the polynucleotide or polypeptideis not found in nature, is sufficiently free of other biologicalmacromolecules so that the polynucleotide or polypeptide can be used forits intended purpose.

Fusion Proteins

The GAS antigens used in the invention may be present in the compositionas individual separate polypeptides, but it is preferred that at leasttwo (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20) of the antigens are expressed as a single polypeptide chain (a‘hybrid’ polypeptide). Hybrid polypeptides offer two principaladvantages: first, a polypeptide that may be unstable or poorlyexpressed on its own can be assisted by adding a suitable hybrid partnerthat overcomes the problem; second, commercial manufacture is simplifiedas only one expression and purification need be employed in order toproduce two polypeptides which are both antigenically useful.

The hybrid polypeptide may comprise two or more polypeptide sequencesfrom the first antigen group. Accordingly, the invention includes acomposition comprising a first amino acid sequence and a second aminoacid sequence, wherein said first and second amino acid sequences areselected from a GAS antigen or a fragment thereof of the first antigengroup. Preferably, the first and second amino acid sequences in thehybrid polypeptide comprise different epitopes.

The hybrid polypeptide may comprise one or more polypeptide sequencesfrom the first antigen group and one or more polypeptide sequences fromthe second antigen group. Accordingly, the invention includes acomposition comprising a first amino acid sequence and a second aminoacid sequence, said first amino acid sequence selected from a GASantigen or a fragment thereof from the first antigen group and saidsecond amino acid sequence selected from a GAS antigen or a fragmentthereof from the second antigen group. Preferably, the first and secondamino acid sequences in the hybrid polypeptide comprise differentepitopes.

Hybrids consisting of amino acid sequences from two, three, four, five,six, seven, eight, nine, or ten GAS antigens are preferred. Inparticular, hybrids consisting of amino acid sequences from two, three,four, or five GAS antigens are preferred.

Different hybrid polypeptides may be mixed together in a singleformulation. Within such combinations, a GAS antigen may be present inmore than one hybrid polypeptide and/or as a non-hybrid polypeptide. Itis preferred, however, that an antigen is present either as a hybrid oras a non-hybrid, but not as both.

Hybrid polypeptides can be represented by the formulaNH₂-A-{-X-L-}_(n)-B-COOH, wherein: X is an amino acid sequence of a GASantigen or a fragment thereof from the first antigen group or the secondantigen group; L is an optional linker amino acid sequence; A is anoptional N-terminal amino acid sequence; B is an optional C-terminalamino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14or 15.

If a —X— moiety has a leader peptide sequence in its wild-type form,this may be included or omitted in the hybrid protein. In someembodiments, the leader peptides will be deleted except for that of the—X-moiety located at the N-terminus of the hybrid protein i.e. theleader peptide of X₁ will be retained, but the leader peptides of X₂ . .. X_(n) will be omitted. This is equivalent to deleting all leaderpeptides and using the leader peptide of X₁ as moiety -A-.

For each n instances of {—X-L-}, linker amino acid sequence -L- may bepresent or absent. For instance, when n=2 the hybrid may beNH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁—X₂—COOH, NH₂—X₁-L₁-X₂—COOHNH₂—X₁—X₂-L₂-COOH, etc. Linker amino acid sequence(s) -L- will typicallybe short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short peptidesequences which facilitate cloning, poly-glycine linkers (i.e.comprising Gly, where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), andhistidine tags (i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more).Other suitable linker amino acid sequences will be apparent to thoseskilled in the art. A useful linker is GSGGGG, with the Gly-Serdipeptide being formed from a BamHI restriction site, thus aidingcloning and manipulation, and the (Gly)₄ tetrapeptide being a typicalpoly-glycine linker.

-A- is an optional N-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leadersequences to direct protein trafficking, or short peptide sequenceswhich facilitate cloning or purification (e.g. histidine tags i.e.His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitableN-terminal amino acid sequences will be apparent to those skilled in theart. If X₁ lacks its own N-terminus methionine, -A- is preferably anoligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) whichprovides a N-terminus methionine.

-B- is an optional C-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includesequences to direct protein trafficking, short peptide sequences whichfacilitate cloning or purification (e.g. comprising histidine tags i.e.H is where n=3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences whichenhance protein stability. Other suitable C-terminal amino acidsequences will be apparent to those skilled in the art.

Most preferably, n is 2 or 3.

The fusion constructs of the invention may include a combination of twoor more GAS antigens, wherein said combination includes GAS 40 or afragment thereof or a polypeptide having sequence identity thereto.

The fusion constructs of the invention may include a combination of GASantigens, said combination consisting of two to thirty-one GAS antigensof the first antigen group, said first antigen group consisting of: GAS117, GAS 130, GAS 277, GAS 236, GAS 40, GAS 389, GAS 504, GAS 509, GAS366, GAS 159, GAS 217, GAS 309, GAS 372, GAS 039, GAS 042, GAS 058, GAS290, GAS 511, GAS 533, GAS 527, GAS 294, GAS 253, GAS 529, GAS 045, GAS095, GAS 193, GAS 137, GAS 084, GAS 384, GAS 202, and GAS 057.Preferably, the combination of GAS antigens consists of three, four,five, six, seven, eight, nine, or ten GAS antigens selected from thefirst antigen group. Preferably, the combination of GAS antigensconsists of three, four, or five GAS antigens selected from the firstantigen group.

GAS 39, GAS 40, GAS 57, GAS 117, GAS 202, GAS 294, GAS 527, GAS 533, andGAS 511 are particularly preferred GAS antigens for use in the fusionconstructs of the invention. Preferably, the combination of GAS antigensincludes either or both of GAS 40 and GAS 117. Preferably, thecombination includes GAS 40.

Recombinant expression of the fusion constructs of the invention may beimproved or optimised by the same methods described for the expressionof the GAS antigens alone (discussed above). Fusion constructs of GAS 40and GAS 117 are exemplified below.

In the first example, GAS 117 is linked to GAS 40a-RR. (As discussedabove, GAS 40a-RR is a codon optimised GAS 40 sequence where theN-terminal leader sequence and the C-terminal transmembrane sequence areremoved). In this construct a GAS 117 fragment (where the N-terminalleader sequence is removed) is placed to the N-terminus of the GAS 40sequence and a HIS tag is added to the C-terminus of the GAS 40sequence. This construct is designated “117-40a-RR” Amino acid andpolynucleotide sequences for this construct are shown in the sequencelisting as SEQ ID NOS: 127 and 128.

The GAS 117 and GAS 40 sequences are preferably linked by a linkersequence comprising multiple Glycine residues. For example, the linkerused in 117-40a-RR fusion construct, a linker sequence of SEQ ID NO: 129(YASGGGS) is used.

In a second example, the relative locations of the GAS 40 and GAS 117sequences can be exchanged. In this construct, designated “40a-RR-117”,the GAS 40a-RR sequence is placed to the N-terminus of the GAS 117sequence and the HIS tag is added to the C-terminus of the GAS 117sequence. Amino acid and polynucleotide sequences for this fusionconstruct are shown in the sequence listing as SEQ ID NOS: 130 and 131.

Alternatively, the fusion constructs may be designed without codonoptimisations. For example, polynucleotide and amino acid sequences forfusion construct “117-40a” is shown in the sequence listing as SEQ IDNOS: 132 and 133. (While no codon optimisations were used, three pointmutations apparently occurred during the cloning, only one of whichinvolved a conservative amino acid change (Glucine to Glycine). In themurine immunization model (previously discussed above), immunizationwith “11740a” has yielded up to 80% survival upon challenge.

A preferred GAS 40 fusion sequence comprises a fragment of GAS 40comprising one or more of the coiled-coil regions. For example, thefusion construct may comprise a GAS 40 sequence comprising the firstcoiled-coil region. “117-40N” is an example of this type of constructAmino acid and polynucleotide sequences for this construct are shown inthe sequence listing as SEQ ID NOS; 132 and 133.

The invention also provides nucleic acids encoding hybrid polypeptidesof the invention. Furthermore, the invention provides nucleic acid whichcan hybridise to this nucleic acid, preferably under “high stringency”conditions (e.g. 65.degree. C. in a 0.1.times.SSC, 0.5% SDS solution).

The GAS antigens of the invention may also be used to prepare antibodiesspecific to the GAS antigens. The antibodies are preferably specific tothe first or second coiled-coil regions of GAS 40. The invention alsoincludes the use of combination of two or more types of antibodiesselected from the group consisting of antibodies specific to GBS 80, GAS117, GAS 130, GAS 277, GAS 236, GAS 40, GAS 389, GAS 504, GAS 509, GAS366, GAS 159, GAS 217, GAS 309, GAS 372, GAS 039, GAS 042, GAS 058, GAS290, GAS 511, GAS 533, GAS 527, GAS 294, GAS 253, GAS 529, GAS 045, GAS095, GAS 193, GAS 137, GAS 084, GAS 384, GAS 202, and GAS 057.Preferably, the combination includes an antibody specific to GAS 40, ora fragment thereof.

The GAS specific antibodies of the invention include one or morebiological moieties that, through chemical or physical means, can bindto or associate with an epitope of a GAS polypeptide. The antibodies ofthe invention include antibodies which specifically bind to a GASantigen, preferably GAS 80. The invention includes antibodies obtainedfrom both polyclonal and monoclonal preparations, as well as thefollowing: hybrid (chimeric) antibody molecules (see, for example,Winter et al. (1991) Nature 349: 293-299; and U.S. Pat. No. 4,816,567;F(ab′)₂ and F(ab) fragments; F_(v) molecules (non-covalent heterodimers,see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096);single-chain Fv molecules (sFv) (see, for example, Huston et al. (1988)Proc Natl Acad Sci USA 85:5897-5883); dimeric and trimeric antibodyfragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem31:1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126);humanized antibody molecules (see, for example, Riechmann et al. (1988)Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; andU.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and,any functional fragments obtained from such molecules, wherein suchfragments retain immunological binding properties of the parent antibodymolecule. The invention further includes antibodies obtained throughnon-conventional processes, such as phage display.

Preferably, the GAS specific antibodies of the invention are monoclonalantibodies. Monoclonal antibodies of the invention include an antibodycomposition having a homogeneous antibody population. Monoclonalantibodies of the invention may be obtained from murine hybridomas, aswell as human monoclonal antibodies obtained using human rather thanmurine hybridomas. See, e.g., Cote, et al. Monoclonal Antibodies andCancer Therapy, Alan R. Liss, 1985, p 77.

Polypeptides of the invention can be prepared by various means (e.g.recombinant expression, purification from cell culture, chemicalsynthesis, etc.) and in various forms (e.g. native, fusions,non-glycosylated, lipidated, etc.). They are preferably prepared insubstantially pure form (i.e. substantially free from other GAS or hostcell proteins).

Nucleic acid according to the invention can be prepared in many ways(e.g. by chemical synthesis, from genomic or cDNA libraries, from theorganism itself, etc.) and can take various forms (e.g. single stranded,double stranded, vectors, probes, etc.). They are preferably prepared insubstantially pure form (i.e. substantially free from other GAS or hostcell nucleic acids).

The term “nucleic acid” includes DNA and RNA, and also their analogues,such as those containing modified backbones (e.g. phosphorothioates,etc.), and also peptide nucleic acids (PNA), etc. The invention includesnucleic acid comprising sequences complementary to those described above(e.g. for antisense or probing purposes).

The invention also provides a process for producing a polypeptide of theinvention, comprising the step of culturing a host cell transformed withnucleic acid of the invention under conditions which induce polypeptideexpression.

The invention provides a process for producing a polypeptide of theinvention, comprising the step of synthesising at least part of thepolypeptide by chemical means.

The invention provides a process for producing nucleic acid of theinvention, comprising the step of amplifying nucleic acid using aprimer-based amplification method (e.g. PCR).

The invention provides a process for producing nucleic acid of theinvention, comprising the step of synthesising at least part of thenucleic acid by chemical means.

Strains

Preferred polypeptides of the invention comprise an amino acid sequencefound in an M1, M3 or M18 strain of GAS. The genomic sequence of an M1GAS strain is reported at Ferretti et al, PNAS (2001) 98(8):4658-4663.The genomic sequence of an M3 GAS strain is reported at Beres et al.,PNAS (2002) 99(15):10078-10083. The genomic sequence of an M18 GASstrain is reported at Smooet et al., PNAS (2002) 99(7):4668-4673.

Where hybrid polypeptides are used, the individual antigens within thehybrid (i.e. individual —X— moieties) may be from one or more strains.Where n=2, for instance, X₂ may be from the same strain as X₁ or from adifferent strain. Where n=3, the strains might be (i) X₁=X₂=X₃ (ii)X₁=X_(≠)X₃ (iii) X_(1≠)X₂=X₃ (iv) X_(1≠)X_(2≠)X₃ or (v) X₁=X_(3≠)X₂,etc.

Purification and Recombinant Expression

The GAS antigens of the invention may be isolated from a Streptococcuspyogenes, or they may be recombinantly produced, for instance, in aheterologous host. Preferably, the GAS antigens are prepared using aheterologous host. The heterologous host may be prokaryotic (e.g. abacterium) or eukaryotic. It is preferably E. coli, but other suitablehosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi,Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea,Mycobacteria (e.g. M. tuberculosis), yeasts, etc.

Recombinant production of polypeptides is facilitated by adding a tagprotein to the GAS antigen to be expressed as a fusion proteincomprising the tag protein and the GAS antigen. Such tag proteins canfacilitate purification, detection and stability of the expressedprotein. Tag proteins suitable for use in the invention include apolyarginine tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag,Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide,cellulose-binding domain, SBP-tag, chitin-binding domain, glutathioneS-transferase-tag (GST), maltose-binding protein, transcriptiontermination anti-termination factor (NusA), E. coli thioredoxin (TrxA)and protein disulfide isomerase I (DsbA). Preferred tag proteins includeHis-tag and GST. A full discussion on the use of tag proteins can befound at Terpe et al., Appl Microbiol Biotechnol (2003) 60:523-533.

After purification, the tag proteins may optionally be removed from theexpressed fusion protein, i.e., by specifically tailored enzymatictreatments known in the art. Commonly used proteases includeenterokinase, tobacco etch virus (TEV), thrombin, and factor X_(a).

Immunogenic Compositions and Medicaments

Compositions of the invention are preferably immunogenic compositions,and are more preferably vaccine compositions. The pH of the compositionis preferably between 6 and 8, preferably about 7. The pH may bemaintained by the use of a buffer. The composition may be sterile and/orpyrogen-free. The composition may be isotonic with respect to humans.

Vaccines according to the invention may either be prophylactic (i.e. toprevent infection) or therapeutic (i.e. to treat infection), but willtypically be prophylactic. Accordingly, the invention includes a methodfor the therapeutic or prophylactic treatment of a Streptococcuspyogenes infection in an animal susceptible to streptococcal infectioncomprising administering to said animal a therapeutic or prophylacticamount of the immunogenic compositions of the invention. Preferably, theimmunogenic composition comprises a combination of GAS antigens, saidcombination consisting of two to thirty-one GAS antigens of the firstantigen group. Preferably, the combination of GAS antigens consists ofthree, four, five, six, seven, eight, nine, or ten GAS antigens selectedfrom the first antigen group. Preferably, the combination of GASantigens consists of three, four, or five GAS antigens selected from thefirst antigen group. Preferably, the combination of GAS antigensincludes either or both of GAS 40 and GAS 117.

Alternatively, the invention includes an immunogenic compositioncomprising a combination of GAS antigens, said combination consisting oftwo to thirty-one GAS antigens of the first antigen group and one, two,three, or four GAS antigens of the second antigen group. Preferably, thecombination consists of three, four, five, six, seven, eight, nine, orten GAS antigens from the first antigen group. Still more preferably,the combination consists of three, four or five GAS antigens from thefirst antigen group. Preferably, the combination of GAS antigensincludes either or both of GAS 40 and GAS 117. Preferably, thecombination of GAS antigens includes one or more variants of the Msurface protein.

The invention also provides a composition of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of the compositions of the inventionin the manufacture of a medicament for raising an immune response in amammal. The medicament is preferably a vaccine. The invention alsoprovides for a kit comprising a first component comprising a combinationof GAS antigens.

In one embodiment, the combination of GAS antigens consists of a mixtureof two to thirty-one GAS antigens selected from the first antigen group.Preferably, the combination consists of three, four, five, six, seven,eight, nine, or ten GAS antigens from the first antigen group.Preferably, the combination consists of three, four, or five GASantigens from the first antigen group. Preferably, the combinationincludes either or both of GAS 117 and GAS 040.

In another embodiment, the kit comprises a first component comprising acombination of GAS antigens consisting of a mixture of two to thirty-oneGAS antigens of the first antigen group and one, two, three, or four GASantigens of the second antigen group. Preferably, the combinationconsists of three, four, five, six, seven, eight, nine, or ten GASantigens from the first antigen group. Still more preferably, thecombination consists of three, four or five GAS antigens from the firstantigen group. Preferably, the combination of GAS antigens includeseither or both of GAS 40 and GAS 117. Preferably, the combination of GASantigens includes one or more variants of the M surface protein.

The invention also provides a delivery device pre-filled with theimmunogenic compositions of the invention.

The invention also provides a method for raising an immune response in amammal comprising the step of administering an effective amount of acomposition of the invention. The immune response is preferablyprotective and preferably involves antibodies and/or cell-mediatedimmunity The method may raise a booster response.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager; where the vaccine is for therapeutic use, the human ispreferably a teenager or an adult. A vaccine intended for children mayalso be administered to adults e.g. to assess safety, dosage,immunogenicity, etc.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by Streptococcus pyogenes (e.g.pharyngitis (such as streptococcal sore throat), scarlet fever,impetigo, erysipelas, cellulitis, septicemia, toxic shock syndrome,necrotizing fasciitis (flesh eating disease) and sequelae (such asrheumatic fever and acute glomerulonephritis)). The compositions mayalso be effective against other streptococcal bacteria.

One way of checking efficacy of therapeutic treatment involvesmonitoring GAS infection after administration of the composition of theinvention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against the GAS antigens in thecompositions of the invention after administration of the composition.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral (e.g.tablet, spray), vaginal, topical, transdermal (e.g. see WO99/27961) ortranscutaneous (e.g. see WO02/074244 and WO02/064162), intranasal (e.g.see WO03/028760), ocular, aural, pulmonary or other mucosaladministration. The invention may be used to elicit systemic and/ormucosal immunity

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. In a multiple dose schedulethe various doses may be given by the same or different routes e.g. aparenteral prime and mucosal boost, a mucosal prime and parenteralboost, etc.

The compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g. a lyophilised composition). The composition may beprepared for topical administration e.g. as an ointment, cream orpowder. The composition may be prepared for oral administration e.g. asa tablet or capsule, as a spray, or as a syrup (optionally flavoured).The composition may be prepared for pulmonary administration e.g. as aninhaler, using a fine powder or a spray. The composition may be preparedas a suppository or pessary. The composition may be prepared for nasal,aural or ocular administration e.g. as drops. The composition may be inkit form, designed such that a combined composition is reconstitutedjust prior to administration to a patient. Such kits may comprise one ormore antigens in liquid form and one or more lyophilised antigens.Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of antigen(s), as well as any other components, asneeded. By ‘immunologically effective amount’, it is meant that theadministration of that amount to an individual, either in a single doseor as part of a series, is effective for treatment or prevention. Thisamount varies depending upon the health and physical condition of theindividual to be treated, age, the taxonomic group of individual to betreated (e.g. non-human primate, primate, etc.), the capacity of theindividual's immune system to synthesise antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials.

Further Components of the Composition

The composition of the invention will typically, in addition to thecomponents mentioned above, comprise one or more ‘pharmaceuticallyacceptable carriers’, which include any carrier that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolised macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and lipid aggregates (such as oil droplets or liposomes). Such carriersare well known to those of ordinary skill in the art. The vaccines mayalso contain diluents, such as water, saline, glycerol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present. Athorough discussion of pharmaceutically acceptable excipients isavailable in Gennaro (2000) Remington: The Science and Practice ofPharmacy. 20th ed., ISBN: 0683306472.

Vaccines of the invention may be administered in conjunction with otherimmunoregulatory agents. In particular, compositions will usuallyinclude an adjuvant.

Preferred further adjuvants include, but are not limited to, one or moreof the following set forth below:

A. Mineral Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. {e.g. see chapters 8 & 9 of Vaccine design: the subunitand adjuvant approach (1995) Powell & Newman. ISBN 0-306-44867-X}), ormixtures of different mineral compounds, with the compounds taking anysuitable form (e.g. gel, crystalline, amorphous, etc.), and withadsorption being preferred. The mineral containing compositions may alsobe formulated as a particle of metal salt. See WO00/23105.

B. Oil-Emulsions

Oil-emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 (5% Squalene, 0.5% TWEEN®80, and 0.5% SPAN® 85, formulated into submicron particles using amicrofluidizer). See WO90/14837. See also, Podda, “The adjuvantedinfluenza vaccines with novel adjuvants: experience with theMF59-adjuvanted vaccine”, Vaccine (2001) 19: 2673-2680; Frey et al.,“Comparison of the safety, tolerability, and immunogenicity of aMF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccinein non-elderly adults”, Vaccine (2003) 21:4234-4237. MF59 is used as theadjuvant in the FLUAD™ influenza virus trivalent subunit vaccine.

Particularly preferred adjuvants for use in the compositions aresubmicron oil-in-water emulsions. Preferred submicron oil-in-wateremulsions for use herein are squalene/water emulsions optionallycontaining varying amounts of MTP-PE, such as a submicron oil-in-wateremulsion containing 4-5% w/v squalene, 0.25-1.0% w/v TWEEN® 80(polyoxyethylenesorbitan monooleate), and/or 0.25-1.0% SPAN® 85(sorbitan trioleate), and, optionally,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphosphoryloxy)-ethylamine(MTP-PE), for example, the submicron oil-in-water emulsion known as“MP59” (International Publication No. WO90/14837; U.S. Pat. Nos.6,299,884 and 6,451,325, incorporated herein by reference in theirentireties; and Ott et al., “MP59—Design and Evaluation of a Safe andPotent Adjuvant for Human Vaccines” in Vaccine Design: The Subunit andAdjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press,New York, 1995, pp. 277-296). MF59 contains 4-5% w/v Squalene (e.g.4.3%), 0.25-0.5% w/v TWEEN® and 0.5% w/v SPAN® 85 and optionallycontains various amounts of MTP-PE, formulated into submicron particlesusing a microfluidizer such as Model 110Y microfluidizer (Microfluidics,Newton, Mass.). For example, MMP-PE may be present in an amount of about0-500 μg/dose, more preferably 0-250 μg/dose and most preferably, 0-100μg/dose. As used herein, the term “MF59-0” refers to the above submicronoil-in-water emulsion lacking MTP-PE, while the term MF59-MTP denotes aformulation that contains MTP-PE. For instance, “M59-100” contains 100μg MTP-PE per dose, and so on. MF69, another submicron oil-in-wateremulsion for use herein, contains 4.3% w/v squalene, 0.25% w/v TWEEN®80, and 0.75% w/v SPAN® 85 and optionally MTP-PE. Yet another submicronoil-in-water emulsion is MF75, also known as SAF, containing 10%squalene, 0.4% TWEEN® 80, 5% pluronic-blocked polymer L121, and thr-MDP,also microfluidized into a submicron emulsion. M5-MTP denotes an M75formulation that includes MTP, such as from 100-400 μg MTP-PE per dose.Submicron oil-in-water emulsions, methods of making the same andimmunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in International Publication No.WO90/14837 and U.S. Pat. Nos. 6,299,884 and 6,451,325, incorporatedherein by reference in their entireties.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)may also be used as adjuvants in the invention.

C. Saponin Formulations

Saponin formulations, may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. Saponin compositions have been purified using High PerformanceThin Layer Chromatography (HP-LC) and Reversed Phase High PerformanceLiquid Chromatography (RP-HPLC). Specific purified fractions using thesetechniques have been identified, including QS7, QS17, QS18, QS21, QH-A,QH-B and QH-C. Preferably, the saponin is QS21. A method of productionof QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulationsmay also comprise a sterol, such as cholesterol (see WO 96/33739).

Combinations of saponins and cholesterols can be used to form uniqueparticles called Immunostimulating Complexes (ISCOMs). ISCOMs typicallyalso include a phospholipid such as phosphatidylethanolamine orphosphatidylcholine. Any known saponin can be used in ISCOMs.Preferably, the ISCOM includes one or more of Quil A, QHA and QHC.ISCOMs are further described in EP 0 109 942, WO 96/11711 and WO96/33739. Optionally, the ISCOMS may be devoid of additional detergent.See WO00107621.

A review of the development of saponin based adjuvants can be found atBarr, et al., Advanced Drug Delivery Reviews (1998) 32:247-271. See alsoSjolander, et al., Advanced Drug Delivery Reviews (1998) 32:321-338.

D. Virosomes and Virus Like Particles (VLPs)

Virosomes and Virus Like Particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein pI). VLPs are discussed furtherin WO 03/024480, WO 03/024481, and Niikura et al., Virology (2002)293:273-280, Lenz et al., Journal of Immunology (2001) 5246-5355; Pinto,et al. Journal of Infectious Diseases (2003) 188:327-338 and Gerber etal., Journal of Virology (2001) 75(10):4752-4760. Virosomes arediscussed further in, for example, Gluck et al., Vaccine (2002)20:B10-B16.

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as:

(1) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylatedMPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipidA with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such“small particles” of 3dMPL are small enough to be sterile filteredthrough a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPSderivatives include monophosphoryl lipid A mimics, such as aminoalkylglucosaminide phosphate derivatives e.g. RC-529. See Johnson et al.(1999) Bioorg Med Chem Lett 9:2273-2278.

(2) Lipid A Derivatives

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as 0M-174. 0M-174 is described for example in Meraldi et al.,Vaccine (2003) 21:2485-2491 and Pajak, et al., Vaccine (2003)21:836-842.

(3) Immunostimulatory Oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (asequence containing an unmethylated cytosine followed by guanosine andlinked by a phosphate bond). Bacterial double stranded RNA oroligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpGs can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. Optionally, the guanosine may be replaced with ananalog such as 2′-deoxy-7-deazaguanosine. See Kandimalla, et al.,Nucleic Acids Research (2003) 31(9): 2393-2400; WO 02/26757 and WO99/62923 for examples of possible analogue substitutions. The adjuvanteffect of CpG oligonucleotides is further discussed in Krieg, NatureMedicine (2003) 9(7): 831-835; McCluskie, et al., FEMS Immunology andMedical Microbiology (2002) 32:179-185; WO 98/40100, U.S. Pat. No.6,207,646, U.S. Pat. No. 6,239,116, and U.S. Pat. No. 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT. See Kandimalla, et al., Biochemical Society Transactions (2003)31 (part 3): 654-658. The CpG sequence may be specific for inducing aTh1 immune response, such as a CpG-A ODN, or it may be more specific forinducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs arediscussed in Blackwell, et al., J. Immunol. (2003) 170(8):4061-4068;Krieg, TRENDS in Immunology (2002) 23(2): 64-65 and WO 01/95935.Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, Kandimalla, et al., BBRC (2003) 306:948-953; Kandimalla, etal., Biochemical Society Transactions (2003) 31(part 3):664-658; Bhagatet al., BBRC (2003) 300:853-861 and WO 03/035836.

(4) ADP-Ribosylating Toxins and Detoxified Derivatives Thereof

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (i.e., E. coli heat labile enterotoxin “LT),cholera (“Cr”), or pertussis (“PT”). The use of detoxifiedADP-ribosylating toxins as mucosal adjuvants is described in WO 95/17211and as parenteral adjuvants in WO 98/42375. Preferably, the adjuvant isa detoxified LT mutant such as LT-K63.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophagecolony stimulating factor, and tumor necrosis factor.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres (Singh et al. (2001) J. Cont. Rele. 70:267-276) ormucoadhesives such as cross-linked derivatives of poly(acrylic acid),polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose. Chitosan and derivatives thereof may also beused as adjuvants in the invention. E.g., WO99/27960.

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

I. Liposomes

Examples of liposome formulations suitable for use as adjuvants aredescribed in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0626 169.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters. WO99/52549. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol (WO01/21207) as well as polyoxyethylene alkyl ethers orester surfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol (WO01/21152).

Preferred polyoxyethylene ethers are selected from the following group:polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steorylether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

K. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in Andrianov et al.,“Preparation of hydrogel microspheres by coacervation of aqueouspolyphophazene solutions”, Biomaterials (1998) 19(1-3):109-115 and Payneet al., “Protein Release from Polyphosphazene Matrices”, Adv. Drug.Delivery Review (1998) 31(3):185-196.

L. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues, described further inStanley, “Imiquimod and the imidazoquinolones: mechanism of action andtherapeutic potential” Clin Exp Dermatol (2002) 27(7):571-577 and Jones,“Resiquimod 3M”, Curr Opin Investig Drugs (2003) 4(2):214-218.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention:

(1) a saponin and an oil-in-water emulsion (WO99/11241);

(2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g., 3dMPL) (seeWO 94/00153);

(3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g., 3dMPL)+acholesterol;

(4) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol)(WO98/57659);

(5) combinations of 3dMPL with, for example, QS21 and/or oil-in-wateremulsions (European patent applications 0835318, 0735898 and 0761231);

(6) SAF, containing 10% Squalane, 0.4% TWEEN® 80, 5% pluronic-blockpolymer L121, and thr-MDP, either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion.

(7) RIBI™ adjuvant system (RAS), (Ribi Immunochem) containing 2%Squalene, 0.2% TWEEN® 80, and one or more bacterial cell wall componentsfrom the group consisting of monophosphorylipid A (MPL), trehalosedimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS(DETOX™); and

(8) one or more mineral salts (such as an aluminum salt)+a non-toxicderivative of LPS (such as 3dPML).

(9) one or more mineral salts (such as an aluminum salt)+animmunostimulatory oligonucleotide (such as a nucleotide sequenceincluding a CpG motif).

Aluminium salts and MF59 are preferred adjuvants for parenteralimmunisation. Mutant bacterial toxins are preferred mucosal adjuvants.

The composition may include an antibiotic.

Further Antigens

The compositions of the invention may further comprise one or moreadditional non-GAS antigens, including additional bacterial, viral orparasitic antigens.

In one embodiment, the GAS antigen combinations of the invention arecombined with one or more additional, non-GAS antigens suitable for usein a paediatric vaccine. For example, the GAS antigen combinations maybe combined with one or more antigens derived from a bacteria or virusselected from the group consisting of N. meningitidis (includingserogroup A, B, C, W135 and/or Y), Streptococcus pneumoniae, Bordetellapertussis, Moraxella catarrhalis, Tetanus, Diphtheria, RespiratorySyncytial virus (‘RSV’), polio, measles, mumps, rubella, and rotavirus.

In another embodiment, the GAS antigen combinations of the invention arecombined with one or more additional, non-GAS antigens suitable for usein a vaccine designed to protect elderly or immunocomprised individuals.For example, the GAS antigen combinations may be combined with anantigen derived from the group consisting of Enterococcus faecalis,Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa,Legionella pneumophila, Listeria monocytogenes, influenza, andParainfluenza virus (‘PIV’).

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier protein in order to enhance immunogenicity {e.g.Ramsay et al. (2001) Lancet 357(9251):195-196; Lindberg (1999) Vaccine17 Suppl 2:S28-36; Buttery & Moxon (2000) J R Coll Physicians Lond34:163-168; Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133,vii. Goldblatt (1998) J. Med. Microbiol. 47:563-567; European patent 0477 508; U.S. Pat. No. 5,306,492; WO98/42721; Conjugate Vaccines (eds.Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114; Hermanson(1996) Bioconjugate Techniques ISBN: 0123423368 or 012342335X}.Preferred carrier proteins are bacterial toxins or toxoids, such asdiphtheria or tetanus toxoids. The CRM₁₉₇ diphtheria toxoid isparticularly preferred {Research Disclosure, 453077 (January 2002)}.Other carrier polypeptides include the N. meningitidis outer membraneprotein {EP-A-0372501}, synthetic peptides {EP-A-0378881 andEP-A-0427347}, heat shock proteins {WO93/17712 and WO94/03208},pertussis proteins {WO98/58668 and EP-A-0471177}, protein D from H.influenzae {WO00/56360}, cytokines {WO91/01146}, lymphokines, hormones,growth factors, toxin A or B from C. difficile {WO00/ 617 61},iron-uptake proteins {W001/72337}, etc. Where a mixture comprisescapsular saccharides from both serogroups A and C, it may be preferredthat the ratio (w/w) of MenA saccharide:MenC saccharide is greater than1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Different saccharides canbe conjugated to the same or different type of carrier protein. Anysuitable conjugation reaction can be used, with any suitable linkerwhere necessary.

Toxic protein antigens may be detoxified where necessary e.g.detoxification of pertussis toxin by chemical and/or genetic means.

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens.

Antigens in the composition will typically be present at a concentrationof at least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using protein antigens in the composition of theinvention, nucleic acid encoding the antigen may be used {e.g. Robinson& Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997)Annu Rev Immunol 15:617-648; Scott-Taylor & Dalgleish (2000) Expert OpinInvestig Drugs 9:471-480; Apostolopoulos & Plebanski (2000) Curr OpinMol Ther 2:441-447; Ilan (1999) Curr Opin Mol Ther 1:116-12 Dubensky etal. (2000) Mol Med 6:723-732; Robinson & Pertmer (2000) Adv Virus Res55:1-74 Donnelly et al. (2000) Am J Respir Crit. Care Med 162(4 Pt2):S190-193 Davis (1999) Mt. Sinai J. Med. 66:84-90}. Protein componentsof the compositions of the invention may thus be replaced by nucleicacid (preferably DNA e.g. in the form of a plasmid) that encodes theprotein.

DEFINITIONS

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 ofCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30. A preferred alignment is determined by theSmith-Waterman homology search algorithm using an affine gap search witha gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrixof 62. The Smith-Waterman homology search algorithm is disclosed inSmith & Waterman (1981) Adv. Appl. Math. 2: 482-489. Similar sequenceidentity methods can be used to determine sequence homology between twopolynucleotide sequences.

The following example demonstrates one way of preparing recombinant GASantigens of the invention and testing their efficacy in a murine model.

Example 1 Preparation of Recombinant GAS Antigens of the Invention andDemonstration of Efficacy in Murine Model

Recombinant GAS proteins corresponding to two or more of the GASantigens of the first antigen group are expressed as follows.

1. Cloning of GAS Antigens for Expression in E. coli

The selected GAS antigens were cloned in such a way to obtain twodifferent kinds of recombinant proteins: (1) proteins having anhexa-histidine tag at the carboxy-terminus (Gas-His) and (2) proteinshaving the hexa-histidine tag at the carboxy-terminus and GST at theamino-terminus (Gst-Gas-His). Type (1) proteins were obtained by cloningin a pET21b+vector (available from Novagen). The type (2) proteins wereobtained by cloning in a pGEX-NNH vector. This cloning strategy allowedfor the GAS genomic DNA to be used to amplify the selected genes by PCR,to perform a single restriction enzyme digestion of the PCR products andto clone then simultaneously into both vectors.

(a) Construction of pGEX-NNH Expression Vectors

Two pairs of complementary oligodeoxyribonucleotides are synthesisedusing the DNA synthesiser ABI394 (Perkin Elmer) and reagents fromCruachem (Glasgow, Scotland). Equimolar amounts of the oligo pairs (50ng each oligo) are annealed in T4 DNA ligase buffer (New EnglandBiolabs) for 10 mm in a final volume of 50 μl and then left to coolslowly at room temperature. With the described procedure the followingDNA linkers are obtained:

gexNN linker (SEQ ID NOS: 137 and 138)      NdeI NheI XmaI   EcoRI   NcoI       SalI     XhoI      SacIGATCCCATATGGCTAGCCCGGGGAATTCGTCCATGGAGTGAGTCGACTGACTCGAGTGATCGAGCTC    GGTATACCGATCGGGCCCCTTAAGCAGGTACCTCACTCAGCTGACTGAGCTCACTAGCTCGAG      NotI CTGAGCGGCCGCATGAA GACTCGCCGGCGTACTTTCGA gexNNH linker(SEQ ID NOS: 139 and 140)      HindIII NotI   XhoI     Hexa-HistidineTCGACAAGCTTGCGGCCGCACTCGAGCATCACCATCACCATCACTGATGTTCGAACGCCGGCGTGAGCACGTAGAGGTAGTGGTAGTGACTATCGA

The plasmid pGEX-KG [K. L. Guan and J. E. Dixon, Anal. Biochem. 192, 262(1991)] is digested with BamHI and HindIII and 100 ng is ligatedovernight at 16.degree. C. to the linker gexNN with a molar ratio of 3:1linker/plasmid using 200 units of T4 DNA ligase (New england Biolabs).After transformation of the ligation product in E. coli DHS, a clonecontaining the pGEX-NN plasmid, having the correct linker, is selectedby means of restriction enzyme analysis and DNA sequencing. The newplasmid pGEX-NN is digested with SalI and HindIII and ligated to thelinker gexNNH. After transformation of the ligation product in E. coliDH5, a clone containing the pGEX-NNH plasmid, having the correct linker,is selected by means of restriction enzyme analysis and DNA sequencing.

(b) Chromosomal DNA Preparation

GAS SF370 strain is grown in THY medium until OD.sub.600 is 0.6-0.8.Bacteria are then centrifuged, suspended in TES buffer with lysozyme (10mg/ml) and mutanolysine (10 U/μl) and incubated 1 hr at 37° C.

Following treatment of the bacterial suspension with RNAase, ProteinaseK and 10% Sarcosyl/EDTA, protein extraction with saturated phenol andphenol/chloroform is carried out. The resulting supernatant isprecipitated with Sodium Acetate/Ethanol and the extracted DNA ispelletted by centrifugation, suspended in Tris buffer and kept at −20°C.

(c) Oligonucleotide Design

Synthetic oligonucleotide primers are designed on the basis of thecoding sequence of each GAS antigen using the sequence of Streptococcuspyogenes SF370 M1 strain. Any predicted signal peptide is omitted, bydeducing the 5′ end amplification primer sequence immediately downstreamfrom the predicted leader sequence. For most GAS antigens, the 5′ tailof the primers (see Table 1, below) include only one restriction enzymerecognition site (NdeI or NheI, or SpeI depending on the gene's ownrestriction pattern); the 3′ primer tails (see Table 1) include a XhoIor a NotI or a HindIII restriction site.

TABLE 1 Oligonucleotide tails of the primers used toamplify genes encoding selected GAS antigens. 5′ tails 3′ tails NdeI 5′GTGCGTCATATG 3′ XhoI 5′ GCGTCTGAG 3′

NheI 5′ GTGCGTGCTAGC 3′ NotI 5′ ACTCGCTAGCGGCCGC 3′

SpeI 5′ GTGCGTACTAGT 3′ HindIII 5′ GCGTAAGCTT 3′

indicates data missing or illegible when filed

As well as containing the restriction enzyme recognition sequences, theprimers include nucleotides which hybridize to the sequence to beamplified. The number of hybridizing nucleotides depends on the meltingtemperature of the primers which can be determined as described[(Breslauer et al., Proc. Nat. Acad. Sci. 83, 3746-50 (1986)]. Theaverage melting temperature of the selected oligos is 50-55° C. for thehybridizing region alone and 65-75° C. for the whole oligos. Oligos canbe purchased from MWG-Biotech S.p.A. (Firenze, Italy).

(d) PCR Amplification

The standard PCR protocol is as follows: 50 ng genomic DNA are used astemplate in the presence of 0.2 μM each primer, 200 μM each dNTP, 1.5 mMMgCl₂, 1×PCR buffer minus Mg (Gibco-BRL), and 2 units of Taq DNApolymerase (Platinum Taq, Gibco-BRL) in a final volume of 100 μl. Eachsample undergoes a double-step amplification: the first 5 cycles areperformed using as the hybridizing temperature of one of the oligosexcluding the restriction enzyme tail, followed by 25 cycles performedaccording to the hybridization temperature of the whole length primers.The standard cycles are as follows:

one cycle:

denaturation: 94° C., 2 min,

5 cycles:

denaturation: 94° C., 30 seconds,

hybridization: 51° C., 50 seconds,

elongation: 72° C., 1 min or 2 mM and 40 sec,

25 cycles:

denaturation: 94° C., 30 seconds,

hybridization: 70° C., 50 seconds,

elongation: 72° C., 1 min or 2 mM and 40 sec,

72° C., 7 min,

4° C.

The elongation time is 1 min for GAS antigens encoded by ORFs shorterthan 2000 bp, and 2 min and 40 seconds for ORFs longer than 2000 bp. Theamplifications are performed using a Gene Amp PCR system 9600 (PerkinElmer).

To check the amplification results, 4 μl of each PCR product is loadedonto 1-1.5 agarose gel and the size of amplified fragments compared withDNA molecular weight standards (DNA markers III or IX, Roche). The PCRproducts are loaded on agarose gel and after electrophoresis the rightsize bands are excised from the gel. The DNA is purified from theagarose using the Gel Extraction Kit (Qiagen) following the instructionof the manufacturer. The final elution volume of the DNA is 50 μl TE (10mM Tris-HCl, 1 mM EDTA, pH 8). One μl of each purified DNA is loadedonto agarose gel to evaluate the yield.

(e) Digestion of PCR Fragments

One-two μg of purified PCR products are double digested overnight at 37°C. with the appropriate restriction enzymes (60 units of each enzyme)using the appropriate restriction buffer in 100 μl final volume. Therestriction enzymes and the digestion buffers are from New EnglandBiolabs. After purification of the digested DNA (PCR purification Kit,Qiagen) and elution with 30 μl TE, 1 μl is subjected to agarose gelelectrophoresis to evaluate the yield in comparison to titratedmolecular weight standards (DNA markers III or IX, Roche).

(f) Digestion of the Cloning Vectors (pET21b+ and pGEX-NNH)

10 μg of plasmid is double digested with 100 units of each restrictionenzyme in 400 μl reaction volume in the presence of appropriate bufferby overnight incubation at 37° C. After electrophoresis on a 1% agarosegel, the band corresponding to the digested vector is purified from thegel using the Qiagen Qiaex II Gel Extraction Kit and the DNA was elutedwith 50 μl TE. The DNA concentration is evaluated by measuring OD₂₆₀ ofthe sample.

(g) Cloning of the PCR Products

Seventy five ng of the appropriately digested and purified vectors andthe digested and purified fragments corresponding to each selected GASantigen are ligated in final volumes of 10-20 μl with a molar ratio of1:1 fragment/vector, using 400 units T4 DNA ligase (New England Biolabs)in the presence of the buffer supplied by the manufacturer. Thereactions are incubated overnight at 16° C.

Transformation of E. coli BL21 (Novagen) and E. coli BL21-DE3 (Novagen)electrocompetent cells is performed using pGEX-NNH ligations and pET21b+ligations respectively. The transformation procedure is as follows: 1-2μL₁ the ligation reaction is mixed with 50 μL₁ of ice cold competentcells, then the cells are poured in a gene pulser 0.1 cm electrodecuvette (Biorad). After pulsing the cells in a MicroPulserelectroporator (Biorad) following the manufacturer instructions thecells are suspended in 0.95 ml of SOC medium and incubated for 45 min at37° C. under shaking. 100 and 900 μl of cell suspensions are plated onseparate plates of agar LB 100 μg/ml Ampicillin and the plates areincubated overnight at 37° C. The screening of the transformants is doneby PCR: randomly chosen transformants are picked and suspended in 30 μlof PCR reaction mix containing the PCR buffer, the 4 dNTPs, 1.5 mMMgCl₂, Taq polymerase and appropriate forward and reverseoligonucleotide primers that are able to hybridize upstream anddownstream from the polylinker of pET21b+ or pGEX-NNH vectors. After 30cycles of PCR, 5 μl of the resulting products are run on agarose gelelectrophoresis in order to select for positive clones from which theexpected PCR band is obtained. PCR positive clones are chosen on thebasis of the correct size of the PCR product, as evaluated by comparisonwith appropriate molecular weight markers (DNA markers III or IX,Roche).

2. Protein Expression

PCR positive colonies are inoculated in 3 ml LB 100 μg/ml Ampicillin andgrown at 37° C. overnight. 70 μL₁ of the overnight culture is inoculatedin 2 ml LB/Amp and grown at 37.degree. C. until OD₆₀₀ of the pET clonesreached the 0.4-0.8 value or until OD₆₀₀ of the pGEX clones reached the0.8-1 value. Protein expression is then induced by adding 1 mM IPTG(Isopropyl 13-D thio-galacto-piranoside) to the mini-cultures. After 3hours incubation at 37° C. the final OD₆₀₀ is checked and the culturesare cooled on ice. After centrifugation of 0.5 ml culture, the cellpellet is suspended in 50 μl of protein Loading Sample Buffer (60 mMTris HCl pH 6.8, 5% w/v SDS, 10% w/v glycerin, 0.1% w/v BromophenolBlue, 100 mM DTT) and incubated at 100° C. for 5 min. A volume of boiledsample corresponding to 0.1 OD₆₀₀ culture is analysed by SDS-PAGE andCoomassie Blue staining to verify the presence of induced protein band.

3. Purification of the Recombinant Proteins

Single colonies are inoculated in 25 ml LB 100 μg/ml Ampicillin andgrown at 37° C. overnight. The overnight culture is inoculated in 500 mlLB/Amp and grown under shaking at 25° C. until OD₆₀₀ 0.4-0.7. Proteinexpression is then induced by adding 1 mM IPTG to the cultures. After3.5 hours incubation at 25. ° C. the final OD₆₀₀ is checked and thecultures are cooled on ice. After centrifugation at 6000 rpm (JA10rotor, Beckman), the cell pellet is processed for purification or frozenat −20° C.

(a) Procedure for the Purification of Soluble His-Tagged Proteins fromE. coli

(1) Transfer the pellets from −20° C. to ice bath and reconstitute with10 ml 50 mM NaHPO₄ buffer, 300 mM NaCl, pH 8.0, pass in 40-50 mlcentrifugation tubes and break the cells as per the following outline.

(2) Break the pellets in the French Press performing three passages within-line washing.

(3) Centrifuge at about 30-40000×g per 15-20 min. If possible use rotorJA 25.50 (21000 rpm, 15 min.) or JA-20 (18000 rpm, 15 min.)

(4) Equilibrate the Poly-Prep columns with 1 ml Fast Flow ChelatingSEPHAROSE® resin with 50 mM phosphate buffer, 300 mM NaCl, pH 80.

(5) Store the centrifugation pellet at −20° C., and load the supernatantin the columns.

(6) Collect the flow through.

(7) Wash the columns with 10 ml (2 ml+2 ml+4 ml) 50 mM phosphate buffer,300 mM NaCl, pH 8.0.

(8) Wash again with 10 ml 20 mM imidazole buffer, 50 mM phosphate, 300mM NaCl, pH 8.0.

(9) Elute the proteins bound to the columns with 4.5 ml (1.5 ml+1.5ml+1.5 ml) 250 mM imidazole buffer, 50 mM phosphate, 300 mM NaCl, pH 8.0and collect the 3 corresponding fractions of 1.5 ml each. Add to eachtube 15 μl DTT 200 mM (final concentration 2 mM)

(10) Measure the protein concentration of the first two fractions withthe Bradford method, collect a 10 μg aliquot of proteins from eachsample and analyse by SDS-PAGE. (N.B.: should the sample be too diluted,load 21 μl+7 μl loading buffer).

(11) Store the collected fractions at +4° C. while waiting for theresults of the SDS-PAGE analysis.

(12) For immunisation prepare 4-5 aliquots of 100 μg each in 0.5 ml in40% glycerol. The dilution buffer is the above elution buffer, plus 2 mMDTT. Store the aliquots at −20° C. until immunisation.

(b) Purification of His-Tagged Proteins from Inclusion Bodies

Purifications are carried out essentially according the followingprotocol:

(1) Bacteria are collected from 500 ml cultures by centrifugation. Ifrequired store bacterial pellets at −20° C. For extraction, resuspendeach bacterial pellet in 10 ml 50 mM Tris —HCl buffer, pH 8.5 on an icebath.

(2) Disrupt the resuspended bacteria with a French Press, performing twopassages.

(3) Centrifuge at 35000×g for 15 min and collect the pellets. Use aBeckman rotor JA 25.50 (21000 rpm, 15 min.) or JA-20 (18000 rpm, 15min.).

(4) Dissolve the centrifugation pellets with 50 mM Tris HCl, 1 mM TCEP(Tris(2-carboxyethyl)-phosphine hydrochloride, Pierce), 6M guanidiumchloride, pH 8.5. Stir for ˜10 min. with a magnetic bar.

(5) Centrifuge as described above, and collect the supernatant.

(6) Prepare an adequate number of Poly-Prep (Bio-Rad) columns containing1 ml of Fast Flow Chelating SEPHAROSE® (Pharmacia) saturated with Nichelaccording to manufacturer recommendations. Wash the columns twice with 5ml of H₂O and equilibrate with 50 mM Tris HCl, 1 mM TCEP, 6M guanidiniumchloride, pH 8.5.

(7) Load the supernatants from step 5 onto the columns, and wash with 5ml of 50 mM Tris HCl buffer, 1 mM TCEP, 6M urea, pH 8.5

(8) Wash the columns with 10 ml of 20 mM imidazole, 50 mM TRIS-HCl, 6Murea, 1 mM TCEP, pH 8.5. Collect and set aside the first 5 ml forpossible further controls.

(9) Elute the proteins bound to the columns with 4.5 ml of a buffercontaining 250 mM imidazole, 50 mM Tris HCl, 6M urea, 1 mM TCEP, pH 8.5.Add the elution buffer in three 1.5 ml aliquots, and collect thecorresponding 3 fractions. Add to each fraction 15 μl DTT (finalconcentration 2 mM).

(10) Measure eluted protein concentration with the Bradford method, andanalyse aliquots of ca 10 μgof protein by SDS-PAGE.

(11) Store proteins at −20° C. in 40% (v/v) glycerol, 50 mM Tris HCl, 2Murea, 0.5 M arginine, 2 mM DTT, 0.3 mM TCEP, 83.3 mM imidazole, pH 8.5.

(c) Procedure for the Purification of GST-Fusion Proteins from E. coli

(1) Transfer the bacterial pellets from −20.degree. C. to an ice bathand suspend with 7.5 ml PBS, pH 7.4 to which a mixture of proteaseinhibitors (COMPLETE™-Boehringer Mannheim, 1 tablet every 25 ml ofbuffer) has been added.

(2) Transfer to 40-50 ml centrifugation tubes and sonicate according tothe following procedure:

a. Position the probe at about 0.5 cm from the bottom of the tube

b. Block the tube with the clamp

c. Dip the tube in an ice bath

d. Set the sonicator as follows: Timer→Hold, Duty Cycle→55, Out.Control→>6.

e. perform 5 cycles of 10 impulses at a time lapse of 1 minute (i.e. onecycle=10 impulses+˜45″ hold; b. 10 impulses+˜45″ hold; c. 10impulses+˜45″ hold; d. 10 impulses+˜45″ hold; e. 10 impulses+˜45″ hold).

(3) Centrifuge at about 30-40000×g for 15-20 min. E.g.: use rotorBeckman JA 25.50 at 21000 rpm, for 15 min.

(4) Store the centrifugation pellets at −20° C., and load thesupernatants on the chromatography columns, as follows

(5) Equilibrate the Poly-Prep (Bio-Rad) columns with 0.5 ml (˜=1 mlsuspension) of Glutathione-SEPHAROSE® 4B resin, wash with 2 ml (1+1)H₂O, and then with 10 ml (2+4+4) PBS, pH 7.4.

(6) Load the supernatants on the columns and discard the flow through.

(7) Wash the columns with 10 ml (2+4+4) PBS, pH 7.4.

(8) Elute the proteins bound to the columns with 4.5 ml of 50 mM Trisbuffer, 10 mM reduced glutathione, pH 8.0, adding 1.5 ml+1.5 ml+1.5 mland collecting the respective 3 fractions of ˜1.5 ml each.

(9) Measure the protein concentration of the first two fractions withthe Bradford method, analyse a 10 μg aliquot of proteins from eachsample by SDS-PAGE. (N.B.: if the sample is too diluted load 21 μl (+7μl loading buffer).

(10) Store the collected fractions at +4° C. while waiting for theresults of the SDS-PAGE analysis.

(11) For each protein destined to the immunisation prepare 4-5 aliquotsof 100 μg each in 0.5 ml of 40% glycerol. The dilution buffer is 50 mMTris HCl, 2 mM DTT, pH 8.0. Store the aliquots at −20° C. untilimmunisation.

4. Murine Model of Protection from GAS Infection

(a) Immunization Protocol

Groups of 10 CD 1 female mice aged between 6 and 7 weeks are immunizedwith two or more GAS antigens of the invention, (20 μg of eachrecombinant GAS antigen), suspended in 100 μl of suitable solution. Eachgroup receives 3 doses at days 0, 21 and 45 Immunization is performedthrough intraperitoneal injection of the protein with an equal volume ofComplete Freund's Adjuvant (CFA) for the first dose and IncompleteFreund's Adjuvant (IFA) for the following two doses. In eachimmunization scheme negative and positive control groups are used.

For the negative control group, mice are immunized with E. coli proteinseluted from the purification columns following processing of totalbacterial extract from a E. coli strain containing either the pET21b orthe pGEX-NNH vector (thus expressing GST only) without any cloned GASORF (groups can be indicated as H is Stop or GSTStop respectively).

For the positive control groups, mice are immunized with purified GAS Mcloned from either GAS SF370 or GAS DSM 2071 strains (groups indicatedas 192SP and 192DSM respectively).

Pooled sera from each group is collected before the first immunizationand two weeks after the last one. Mice are infected with GAS about aweek after.

Immunized mice are infected using a GAS strain different from that usedfor the cloning of the selected proteins. For example, the GAS straincan be DSM 2071 M23 type, obtainable from the German Collection ofMicroorganisms and Cell Cultures (DSMZ).

For infection experiments, DSM 2071 is grown at 37° C. in THY brothuntil OD₆₀₀ 0.4. Bacteria are pelletted by centrifugation, washed oncewith PBS, suspended and diluted with PBS to obtain the appropriateconcentration of bacteria/ml and administered to mice by intraperitonealinjection. Between 50 and 100 bacteria are given to each mouse, asdetermined by plating aliquots of the bacterial suspension on 5 THYplates. Animals are observed daily and checked for survival.

5. Analysis of Immune Sera

(a) Preparation of GAS Total Protein Extracts

Total protein extracts are prepared by incubating a bacterial culturegrown to OD₆₀₀ 0.4-0.5 in Tris 50 mM pH 6.8/mutanolysin (20 units/ml)for 2 hr at 37° C., followed by incubation for ten minutes on ice in0.24 N NaOH and 0.96% (3-mercaptoethanol. The extracted proteins areprecipitated by addition of trichloroacetic acid, washed with ice-coldacetone and suspended in protein loading buffer.

(b) Western Blot Analysis

Aliquots of total protein extract mixed with SDS loading buffer (lx: 60mM Tris HCl HCl pH 6.8, 5% w/v SDS, 10% v/v glycerin, 0.1% BromophenolBlue, 100 mM DTT) and boiled 5 minutes at 95° C., were loaded on a 12.5%SDS-PAGE precast gel (Biorad). The gel is run using a SDS-PAGE runningbuffer containing 250 mM Tris, 2.5 mM Glycine and 0.1% SDS. The gel iselectroblotted onto nitrocellulose membrane at 200 mA for 60 minutes.The membrane is blocked for 60 minutes with PBS/0.05% TWEEN® 20 (Sigma),10% skimmed milk powder and incubated O/N at 4° C. with PBS/0.05% TWEEN®20, 1% skimmed milk powder, with the appropriate dilution of the sera.After washing twice with PBS/0.05% TWEEN®, the membrane is incubated for2 hours with peroxidase-conjugated secondary anti-mouse antibody(Amersham) diluted 1:4000. The nitrocellulose is washed three times for10 minutes with PBS/0.05% TWEEN® and once with PBS and thereafterdeveloped by Opti-4CN Substrate Kit (Biorad).

(c) Preparation of Paraformaldehyde Treated GAS Cultures

A bacterial culture grown to OD₆₀₀ 0.4-0.5 is washed once with PBS andconcentrated four times in PBS/0.05% Paraformaldehyde. Following 1 hrincubation at 37° C. with shacking, the treated culture is keptovernight at 4° C. and complete inactivation of bacteria is thencontrolled by plating aliquots on THY blood agar plates.

(d) FACS analysis of Paraformaldehyde Treated GAS Cultures with MouseImmune Sera

About 10⁵ Paraformaldehyde inactivated bacteria are washed with 200 μlof PBS in a 96 wells U bottom plate and centrifuged for 10 min. at 3000g, at 4° C. The supernatant is discarded and the bacteria are suspendedin 20 μl of PBS-0.1% BSA. Eighty μl of either pre-immune or immune mousesera diluted in PBS-0.1% BSA are added to the bacterial suspension to afinal dilution of either 1:100, 1:250 or 1:500, and incubated on ice for30 min. Bacteria are washed once by adding 100 μL₁ of PBS-0.1% BSA,centrifuged for 10 min. at 3000 g, 4° C., suspended in 200 μl ofPBS-0.1% BSA, centrifuged again and suspended in 10 μl of GoatAnti-Mouse IgG, F(ab′)₂ fragment specific-R-Phycoerythrin-conjugated(Jackson Immunoresearch Laboratories Inc., cat. N_(o) 115-116-072) inPBS-0.1% BSA to a final dilution of 1:100, and incubated on ice for 30min. in the dark. Bacteria are washed once by adding 180 μl of PBS-0.1%BSA and centrifuged for 10 min. at 3000 g, 4.degree. C. The supernatantis discarded and the bacteria were suspended in 200 μl of PBS. Bacterialsuspension is passed through a cytometric chamber of a FACS Calibur(Becton Dikinson, Mountain View, Calif. USA) and 10,000 events areacquired. Data are analysed using Cell Quest Software (Becton Dikinson,Mountain View, Calif. USA) by drawing a morphological dot plot (usingforward and side scatter parameters) on bacterial signals. An histogramplot is then created on FL2 intensity of fluorescence log scalerecalling the morphological region of bacteria.

Example 2 Comparison of Virulence of Wild Type Gas Strain (Including GAS40) and Gas 40 Deletion Mutant

The following example provides a comparison between the virulence of awild type GAS strain and a GAS 40 deletion mutant. Mutant GAS strainswhere a majority of the GAS 40 sequence is removed were prepared bystandard methods. Immunization groups of ten mice per group wereinjected with either the wild type or mutant GAS strains. As shownbelow, injection of a range of concentrations of the wild type isolateresulted in mouse fatalities, while injection with the GAS 440 mutantdid not.

GAS strain concentration number of fatalities wild type 2 × 10⁵ 10 wildtype 2 × 10⁶ 9 wild type 2 × 10⁷ 5 GASΔ40 2 × 10² 0 GASΔ40 2 × 10³ 0GASΔ40 2 × 10⁴ 0 GASΔ40 2 × 10⁵ 0 GASΔ40 2 × 10⁶ 0 GASΔ40 2 × 10⁷ 0

Example 3 Bacterial Opsonophagocytosis Assay of GAS 40 Constructs

The following example demonstrates the surface exposure of GAS 40 by usein a bacterial opsonophagocytosis assay. The following GAS constructs,each of which is described in detail above, were used in the assay:40a-CH, 40a-RR-NH, 40a-RR, GST-40, 40a, 40a and 40a-NH. (The tworeferences to “40a” in FIG. 7 refer to sera prepared on different days.

The assay was performed as follows.

1. Preparation of bacterial inoculum. GAS bacteria are grown in THYmedium until they reach the middle exponential phase (0D₆₀₀ 0.4) at 37°C. Bacteria are washed twice in chilled saline solution and aresuspended in HBSS medium with the volume being adjusted for each straindepending on the amount of bacteria which will be used. Bacterial cellsare kept in ice until use.2. Preparation of PMN. PMN are prepared from buffy coats of heparinizedblood from healthy volunteers. The buffy coat is incubated for 30minutes in a solution containing dextran, NaCl and Heparin (rate 1:1).After incubation the supernatant, rich of leukocytes, is removed,transferred in a clean tube and centrifuged at 700×g for 20 minutes. Ashort wash in water is performed to break red blood cells and then asolution of NaCl is added to restore the appropriate salt concentration.After this step cells are centrifuged, washed and suspended in MEM at asuitable concentration.3. Opsonophagocvtosis assay. GAS strains (prepared as described) areincubated with heat inactivated immune mice serum derived fromimmunization with the indicated GAS antigen (or preimmune for thecontrol), human PMN and baby rabbit complement. 1 hour of incubation at37° C. Samples taken immediately before and after the incubation areplated on THY blood agar plates. Phagocytosis is evaluated comparing thedifference in the number of colonies at the two times for the preimmuneand the immune serum. Data are reported as logarithm number of growncolonies at t=0-logarithm number of grown colonies at t=60

The results of the assay are shown in FIG. 7. The Y axis reports thedifference between the logarithm of colony counts at time 0 and thelogarithm of the colony counts after 60 seconds:log(CFU@T0)-log(CFU@T60′). If there has been growth (i.e., the bacteriaare not actively killed), negative numbers (negative bars) result. Ifbacteria are killed, positive numbers (positive histogram bars) result.As shown in FIG. 7, positive histogram bars are reported for each of theGAS constructs. The last four yellow bars in FIG. 7 represent controls:B=bacteria alone, B PMN=bacteria+polymorphonucleates, BC=Bacteria+complement, P PMN C=bacteria+polymorphonucleates+complement(no serum).

Example 4 GAS 40 Immunization Challenge Experiments in Murine MouseModel of Protection

A sample of the percent survival results from numerous murine mousemodel experiments using the GAS 40 antigen are listed below. Annotationsindicate where construct used to express the recombinant GAS 40 antigenwas modified to facilitate expression.

GAS antigen % Survival in Mouse Challenge Model 40a 55 40a-RR 7040a-RR-NH 60

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

1. An immunogenic composition comprising a combination of Streptococcuspyogenes (GAS) antigens in substantially pure form, said combinationconsisting of two to ten GAS antigens, wherein said combinationincludes: (A) a Spy0269 antigen which comprises a fragment of 200 ormore consecutive amino acids of SEQ ID NO:1; and (B) a Spy0448 antigenwhich comprises a fragment of 25 or more consecutive amino acids of SEQID NO:35.
 2. The immunogenic composition of claim 1 wherein the Spy0269antigen comprises amino acids 27-849 of SEQ ID NO:1.
 3. The immunogeniccomposition of claim 1 wherein the Spy0269 antigen lacks amino acids2-26 of SEQ ID NO:1.
 4. The immunogenic composition of claim 2 whereinthe Spy0269 antigen lacks amino acids 2-26 of SEQ ID NO:1.
 5. Theimmunogenic composition of claim 1 wherein the Spy0269 antigen lacksamino acids 850-873 of SEQ ID NO:1.
 6. The immunogenic composition ofclaim 2 wherein the Spy0269 antigen lacks amino acids 850-873 of SEQ IDNO:1.
 7. The immunogenic composition of claim 3 wherein the Spy0269antigen lacks amino acids 850-873 of SEQ ID NO:1.
 8. The immunogeniccomposition of claim 1 wherein the Spy0448 antigen comprises SEQ IDNO:38.
 9. The immunogenic composition of claim 1 further comprising anadjuvant.
 10. A fusion construct comprising: (A) a Spy0269 antigen whichcomprises a fragment of 200 or more consecutive amino acids of SEQ IDNO:1; and (B) a Spy0448 antigen which comprises a fragment of 25 or moreconsecutive amino acids of SEQ ID NO:35.
 11. The fusion construct ofclaim 10 wherein the Spy0269 antigen comprises amino acids 27-849 of SEQID NO:1.
 12. The fusion construct of claim 10 wherein the Spy0269antigen lacks amino acids 2-26 of SEQ ID NO:1.
 13. The fusion constructof claim 12 wherein the Spy0269 antigen lacks amino acids 2-26 of SEQ IDNO:1.
 14. The fusion construct of claim 10 wherein the Spy0269 antigenlacks amino acids 850-873 of SEQ ID NO:1.
 15. The fusion construct ofclaim 12 wherein the Spy0269 antigen lacks amino acids 850-873 of SEQ IDNO:1.
 16. The fusion construct of claim 13 wherein the Spy0269 antigenlacks amino acids 850-873 of SEQ ID NO:1.
 17. The fusion construct ofclaim 10 wherein the Spy0448 antigen comprises SEQ ID NO:38.
 18. Thefusion construct of claim 10 wherein the Spy0448 antigen is linked tothe N terminus of the Spy0269 antigen.
 19. The fusion construct of claim10 wherein the Spy0448 antigen is linked to the C terminus. of theSpy0269 antigen.
 20. The fusion construct of claim 10 further comprisingan amino acid linker between the Spy0269 antigen and the Spy0448antigen.
 21. The fusion construct of claim 10 which comprises an aminoacid sequence selected from the group consisting of: amino acids 1-929of SEQ ID NO:128; amino acids 1-929 of SEQ ID NO:131; and amino acids1-929 of SEQ ID NO:133.
 22. An immunogenic composition comprising thefusion construct of claim
 10. 23. The immunogenic composition of claim22 further comprising an adjuvant.
 24. A method of raising an immuneresponse against Streptococcus pyogenes, comprising administering to ananimal in need thereof an effective amount of an immunogenic compositioncomprising: (1) a combination of Streptococcus pyogenes (GAS) antigensin substantially pure form, said combination consisting of two to tenGAS antigens, wherein said combination includes: (A) a Spy0269 antigenwhich comprises a fragment of 200 or more consecutive amino acids of SEQID NO:1; and (B) a Spy0448 antigen which comprises a fragment of 25 ormore consecutive amino acids of SEQ ID NO:35; or (2) fusion constructcomprising: (A) a Spy0269 antigen which comprises a fragment of 200 ormore consecutive amino acids of SEQ ID NO:1; and (B) a Spy0448 antigenwhich comprises a fragment of 25 or more consecutive amino acids of SEQID NO:35.